Inhibitors of the JNK signal transduction pathway and methods of use

ABSTRACT

JNK-interacting protein 1 (JIP-1), an inhibitor of the JNK1 protein, and methods of treating a pathological condition or of preventing the occurrence of a pathological condition in a patient by the administration of a therapeutically effective amount of JIP-1 polypeptides, peptides, peptide mimetics, or nucleic acids are described.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0001] This invention was made, in part, with Government support undergrants CA58396 and CA65831 awarded by the National Cancer Institute. Thegovernment may have certain rights in the invention.

FIELD OF THE INVENTION

[0002] The invention relates to signal transduction inhibitors.

BACKGROUND OF THE INVENTION

[0003] The c-Jun NH2-terminal kinase (JNK) is a member of thestress-activated group of Mitogen Activated Protein kinases (MAPkinases) implicated in the control of cell growth. The JNK signaltransduction pathway is activated in response to environmental stressand by the engagement of several classes of cell surface receptors,including cytokine receptors, serpentine receptors, and receptortyrosine kinases. Whitmarsh et al., J. Mol. Med., 74:589 (1996). Inaddition, genetic studies of Drosophila have demonstrated that JNK isrequired for early embryonic development. Sluss et al., Genes & Dev.,10:2745 (1996); Riesgo-Escovar et al., Genes & Dev. 10:2759 (1996). Inmammalian cells, JNK has been implicated in the immune response,oncogenic transformation, and apoptosis. JNK mediates these effects, atleast in part, by increasing the expression of target genes. Targets ofthe JNK signal transduction pathway include the transcription factorsc-Jun, ATF2, and Elk-1. Whitmarsh et al., supra.

[0004] JNK is activated in the liver by metabolic oxidative stress.Mendelson et al., Proc. Natl. Acad. Sci. USA 93:12908-12913 (1996).Activation of JNK also occurs in the kidney during stress, for example,during Bchemic renal failure. Demari et al., Am. J. Physiol.272:F292-F298 (1997). JNK is also activated during cardiovasculardisease such as ischemia Ireperfusion and during organ transplantation.Pombo et al., J. Biol. Chem. 269:26546-26551 (1994); Force et al., Circ.Res. 78:947-53 (1996).

[0005] While JNK is located in both the cytoplasmic and the nuclearcompartments of quiescent cells, activation of the JNK signaltransduction pathway is associated with accumulation of JNK in thenucleus. Mechanisms governing this sub-cellular distribution have notbeen previously elucidated.

[0006] Anchor or tethering proteins play an important role in theregulation of multiple signal transduction pathways. These anchorproteins, which include the nuclear factor kappa B (NFkB) inhibitor IkB,the A kinase anchor protein (AKAP) group of proteins that bind the typeII cyclic adenosine monophosphate (AMP) dependent protein kinase, andthe p190 protein that binds Ca²⁺-calmodulin-dependent protein kinase II,localize their tethered partners to specific sub-cellular compartments.Verma et al., Genes & Dev., 9:2723 (1995); McNeill et al., J. Biol.Chem., 270:10043 (1995); Faux et al., Trends Biochem. Sci., 21:312(1996)). Anchor proteins also target enzymes to specific substrates, andcreate multi-enzyme signaling complexes, such as the Ste5 MAP kinasescaffold complex and the AKAP79 kinase/phosphatase scaffold complex.Choi et al., Cell, 78:499 (1994); Klauck et al., Science, 271:1589(1996); Faux et al., Cell, 85:9 (1996)).

SUMMARY OF THE INVENTION

[0007] The invention, which is based on the discovery of a cytoplasmicanchor protein, JNK-interacting protein 1 (JIP-1; SEQ ID NO:1), featuresJIP-1 polypeptides and nucleic acids, therapeutic compositionscontaining these polypeptides and nucleic acids, and methods ofadministering these compositions. JIP-1 specifically binds to andinhibits the biological effects of JNK, including the initiation ofapoptosis and oncogenic transformation. JIP-1 is therefore useful as atherapeutic agent for treating pathological conditions characterized byapoptosis or transformation. For example, JIP-1 compositions can be usedto treat neurodegenerative diseases characterizediby apoptosis,including Parkinson's disease and Alzheimer's disease; and blood clots,which left untreated could result in stroke and associated memory loss.Other conditions that can be treated using the compositions and methodsof the invention are autoimmune diseases such as arthritis; otherconditions characterized by inflammation; and malignancies, such asleukemias, e.g., chronic myelogenous leukemia (CML). Other conditionsthat can be treated with JIP-1 compositions include oxidative damage toorgans such as the liver and kidney, and heart disease, particularlydamage due to ischemia/reperfusion and cardiomyopathy. JIP-1compositions can also be used to treat donor organs for transplantation.These organs are exposed to substantial environmental stress, theeffects of which are blocked by JNK inhibitors such as JIP-1.

[0008] The invention features a substantially pure JIP-1 polypeptide. A“JIP-1 polypeptide” is a protein having an amino acid sequence thatspecifically binds JNK to the same extent, or at least 10% of thebinding activity of wildtype JIP-1. Such polypeptides can be from 5 to200 amino acids in length, e.g., from 10 to 100 amino acids in length,or from 20 to 50 amino acids in length. Such polypeptides include theJNK Binding Domain (JBD), or portions thereof, of JIP-1 (e.g., aminoacids 148 to 174, forming the “core” of the JBD of wildtype JIP-1, shownin FIG. 2, and having the sequence SGDTYRPKRPTTLNLFPQVPRSQDTLN; SEQ IDNO:3). JIP-1 polypeptides are preferably derived from a mammal, such asa mouse or a human.

[0009] In various embodiments, the polypeptide is soluble, thepolypeptide includes the JNK-binding domain of JIP-1 or a portionthereof, the polypeptide is at least 80%, 90%, or 100% identical to theamino acid sequence from amino acid 148 to amino acid 174 of JIP-1 (thecore JNK-binding domain; SEQ ID NO:3), or the polypeptide has an aminoacid sequence identical to the amino acid sequence from amino acid 148to amino acid 174 of JIP-1 (SEQ ID NO:3), or the polypeptide is at least80%, 90%, or 100% identical to the amino acid sequence from amino acid127 to amino acid 281 of JIP-1 (the JNK-binding domain; SEQ ID NO:4).

[0010] The polypeptides of the invention can be modified to enhancetheir uptake by cells. Such modifications increase the hydrophobicity ofmolecules to facilitate passage through the lipid bilayer of the cellmembrane. For example, polypeptides can be complexed with myristic acidor packaged in liposomes. Alternatively, JIP-1 polypeptides can becomplexed with hydrophobic moieties (e.g., lipids) or peptides thatincrease the delivery of proteins into cells.

[0011] The invention also includes peptide mimetics of JIP-1polypeptides. A “peptide mimetic” of a known polypeptide is a compoundthat mimics the activity of the peptide or polypeptide, but which iscomposed of molecules other than, or in addition to, amino acids.

[0012] By “polypeptide” is meant any chain of amino acids, regardless oflength or post-translational modification (e.g., glycosylation orphosphorylation), and thus includes peptides, proteins, and fusionproteins.

[0013] A “substantially identical” polypeptide sequence differs from agiven sequence only by conservative amino acid substitutions or by oneor more nonconservative substitutions, deletions, or insertions locatedat positions which do not destroy the function of the polypeptidecompared to wildtype JIP-1. Polypeptides of the invention can be 70%,80%, 85%, 90%, or 95% identical to wildtype JIP-1.

[0014] A “substantially pure” preparation is at least 60% by weight ofthe compound of interest, e.g., a JIP-1 polypeptide or fragment of aJIP-1 polypeptide. Preferably the preparation is at least 75%, morepreferably at least 90%, and more preferably at least 95% by weight ofthe compound of interest. Purity can be measured by any appropriatestandard method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or High Pressure Liquid Chromatography (HPLC) analysis.

[0015] The polypeptides of the invention include, but are not limitedto, recombinant polypeptides, natural polypeptides, and syntheticpolypeptides, as well as preproteins or proproteins and biologicallyactive fragments. A “biologically active fragment” of JIP-1 is afragment having at least 50%, 70%, 80%, 90%, 95%, or 100% or greater, ofthe activity of naturally occurring or synthetic, full length JIP-1.

[0016] The polypeptides of the invention can be physically linked toanother polypeptide, e.g., a marker polypeptide. For example, thepolypeptide can be fused to a hexa-histidine tag to facilitatepurification of bacterially expressed proteins, or a hemagglutinin tagto facilitate purification of protein expressed in eukaryotic cells.

[0017] In another aspect, the invention features an isolated nucleicacid that includes a sequence encoding a JIP-1 polypeptide or a fragmentof such a polypeptide. Preferably, the nucleic acid is derived from amammal.

[0018] The invention also encompasses nucleic acids that hybridize understringent conditions (as described herein) to a nucleic acid encoding aJIP-1 polypeptide. Stringent conditions include hybridization at 68° C.in 5×SSC/5×Denhardt's solution/1.0% SDS, or in 0.5 M NaHPO₄ (pH 7.2)/1mM EDTA/7% SDS, or in 50% formamide/0.25 M NaHPO₄ (pH 7.2)/0.25 M NaCl/1mM EDTA/7% SDS; and washing in 0.2×SSC/0.1% SDS at room temperature orat 42° C., or in 0.1×SSC/0.1% SDS at 68° C., or in 40 mM NaHPO₄ (pH7.2)/1 mM EDTA/5% SDS at 50° C., or in 40 mM NaHPO₄ (pH 7.2) 1 mMEDTA/1% SDS at 50° C. Moderately stringent conditions include washing in3×SSC at 42° C. The parameters of salt concentration and temperature canbe varied to achieve the desired level of identity between the probe andthe target nucleic acid. For guidance regarding such conditions see,e.g., Sambrook et al., Molecular Cloninc, A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.; and Ausubel et al.,supra, at Unit 2.10.

[0019] The hybridizing portion of the hybridizing nucleic acid ispreferably 20, 30, 50, or 70 bases long. The hybridizing portion of thehybridizing nucleic acid can be 95% or even 98% or 100% identical to thesequence of a portion of a nucleic acid encoding a JIP-1 polypeptide.Hybridizing nucleic acids of the type described above can be used as acloning probe, a primer (e.g., a PCR primer), or a diagnostic probe.Preferred hybridizing nucleic acids encode a polypeptide having some orall of the biological activities possessed by naturally-occurring JIP-1.Thus, they may encode a protein that is shorter or longer than thevarious forms of JIP-1 described herein. Hybridizing nucleic acids canalso encode proteins that are related to JIP-1, e.g., proteins encodedby genes that include a portion having a relatively high degree ofidentity to a JIP-1 gene described herein.

[0020] The term “nucleic acid” encompasses both RNA and DNA, includingcDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Thenucleic acid may be double-stranded or single-stranded. Wheresingle-stranded, the nucleic acid may be the sense strand or theantisense strand.

[0021] An “isolated nucleic acid” is a nucleic acid that is free of thenucleic acids that normally flank it in the genome. The term thereforeincludes, e.g., a recombinant nucleic acid incorporated into a vector,such as an autonomously replicating plasmid or virus; a cDNA or genomicDNA fragment produced by polymerase chain reaction (PCR) or restrictionendonuclease treatment; and recombinant DNA which is part of a hybridgene encoding additional polypeptide sequences.

[0022] A “substantially identical” nucleic acid is a nucleic acid with asequence that is at least 50%, preferably 70%, and more preferably 85%,90%, or 95% homologous to a given nucleic acid sequence, e.g., SEQ IDNO:2.

[0023] The invention also features transformed cells harboring a nucleicacid encompassed by the invention. Vectors and plasmids that include anucleic acid properly positioned for expression are also within theinvention. A “transformed cell” is a cell into which (or into anancestor of which) has been introduced, by means of recombinant DNAtechniques, a DNA molecule encoding a JIP-1 polypeptide.

[0024] “Operably linked” means that the selected DNA molecule ispositioned adjacent to one or more sequence elements that directtranscription and/or translation of the sequence such that the sequenceelements can control transcription and/or translation of the selectedDNA (i.e., the selected DNA is operably associated with the sequenceelements). Such operably associated elements can be used to facilitatethe production of a JIP-1 polypeptide.

[0025] The invention also features purified antibodies whichspecifically bind a JIP-1 protein or polypeptide. A “purified antibody”is an antibody which is at least 60%, by dry weight, free from theproteins and naturally-occurring organic molecules with which it isnaturally associated. The preparation can be at least 75%, at least 90%,and up to 99% or more, by dry weight, antibody.

[0026] An antibody that “specifically binds” an antigen recognizes andbinds to that antigen, e.g., a JIP-1 polypeptide.

[0027] Also within the invention are antisense molecules and ribozymesfor inhibiting JIP-1 expression.

[0028] The invention also features antagonists and agonists of JIP-1.Antagonists can inhibit one or more of the functions of JIP-1. Suitableantagonists can include large or small molecules, antibodies to JIP-1,and JIP-1 polypeptides that compete with a native form of JIP-1. Suchantagonists include SEQ ID NO:3, a component of an active site of JIP-1,i.e., the JNK-binding domain. Agonists of JIP-1 will enhance orfacilitate one or more of the functions of JIP-1. Agonists andAntagonists include polyproline motifs, which bind to SH3 domains suchas that found in JIP-1.

[0029] A “therapeutically effective amount” of a substance is an amountcapable of producing a medically desirable result in a treated patient,e.g., inhibition of the expression or activity of a specific protein.

[0030] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the arts of protein chemistry or molecular biology. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described infra. All publications,patent applications, patents and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be limiting.

[0031] Other features and advantages of the invention will be apparentfrom the detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1A is a schematic diagram showing the structure of a murineJIP-1 polypeptide.

[0033]FIG. 1B is a representation of the amino acid sequence of a murineJIP-1 polypeptide (SEQ ID NO:1), presented in single letter code.

[0034]FIG. 1C is a representation of the nucleotide sequence of a murineJIP-1 cDNA (SEQ ID NO:2).

[0035]FIG. 2 is a diagram showing an alignment of the JBDs (JNK-bindingdomains) of JIP-1 (SEQ ID NO:3), c-Jun (SEQ ID NO:5), and ATF2 (SEQ IDNO:6).

[0036]FIG. 3 is a diagram showing the amino acid sequences of wild typeand mutant JIP-1 peptides (SEQ ID NOs:3 and 7 to 11), as well as acontrol peptide (SEQ ID NO:12).

[0037]FIG. 4A is a bar graph showing the effect of recombinant JBD(JIP-1 residues 127-281) on reporter gene expression mediated by theGAL4 binding domain and GAL4 fusions with the c-myc, Sp1, and VP16activation domains.

[0038]FIG. 4B is a bar graph showing the effect of recombinant JBD(JIP-1 residues 127-281) on reporter gene expression mediated by wildtype and mutant forms of GAL4-c-Jun, GAL4-ATF2, and GAL4-Elk-1.

[0039]FIG. 4C is a bar graph showing the effect of wild type and mutantJIP-1 and JED on reporter gene expression mediated by wild type andmutant GAL4-ATF2.

[0040]FIG. 5 is a bar graph showing the effect of the JBD of JIP-1 onNerve Growth Factor (NGF) withdrawal-induced apoptosis.

[0041]FIG. 6 is a bar graph showing the effect of bicistronicretroviruses on primary mouse bone marrow cells.

DETAILED DESCRIPTION

[0042] The invention is based on the molecular cloning andcharacterization of JIP-1, a cytoplasmic protein that specifically bindsJNK. JIP-1 polypeptides cause cytoplasmic retention of JNK andinhibition of JNK-regulated gene expression. In addition, JIP-1polypeptides suppress the effects of the JNK signaling pathway,including oncogenic transformation and apoptosis. These findings haveimportant implications for the treatment or prevention of pathologicalconditions and diseases, many of which are characterized bytransformation or apoptosis. Conditions associated with apoptosisinclude neurodegenerative conditions, such as Parkinson's disease orAlzheimer's disease; and blood clots, which left untreated could resultin stroke and associated memory loss. Other conditions that can betreated using the compositions and methods of the invention areautoimmune diseases such as arthritis; other conditions characterized byinflammation; and malignancies, such as leukemias, e.g., chronicmyelogenous leukemia (CML). JIP-1 polypeptide compositions can also beused to treat oxidative damage to organs such as the liver and kidney.Heart disease can also be treated with the compositions of theinvention. Donor organs for transplantation can also be treated withJIP-1 compositions.

[0043] JIP-1 Proteins and Polypeptides

[0044] As shown in FIG. 1, the 660 amino acid JIP-1 protein has an SH3domain at its carboxy terminal end, at amino acid positions 491-540, anda JNK-binding domain (JBD) at its amino terminal end, at amino acidpositions 127-281 (SEQ ID NO:4). The core of the JBD is amino acids148-174 (SEQ ID NO:3). The JED of JIP-1 shares conserved residues withthe JNK-binding regions of the transcription factors c-Jun and ATF2, asshown in FIG. 2.

[0045] JIP-1 polypeptides can be prepared for a wide range of usesincluding, but not limited to, generation of antibodies, preparation ofreagents for diagnostic assays, identification of other moleculesinvolved in transformation or apoptosis, preparation of reagents for usein screening assays for modulators of apoptosis or transformation, andpreparation of therapeutic agents for treatment of disorders related toapoptosis or transformation.

[0046] The invention encompasses, but is not limited to, JIP-1polypeptides that are functionally related to JIP-1 encoded by thenucleotide sequence of FIG. 1C (SEQ ID NO:2) Functionally relatedpolypeptides include any polypeptide sharing a functional characteristicwith wildtype JIP-1 protein, e.g., the ability to bind JNK polypeptidesor to affect proliferation or apoptosis. Such functionally related JIP-1polypeptides include, but are not limited to, polypeptides havingadditions or substitutions of amino acid residues within the amino acidsequence encoded by the JIP-1 sequences described herein which result ina silent change, thus producing a functionally equivalent gene product.Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved.

[0047] For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

[0048] While random mutations can be made to JIP-1 DNA (using randommutagenesis techniques well known to those skilled in the art) and theresulting mutant JIP-1 proteins can be tested for activity,site-directed mutations of the JIP-1 coding sequence can be engineered(using site-directed mutagenesis techniques well known to those skilledin the art) to generate mutant JIP-1 proteins with increased function,e.g., greater JNK-1 binding and inhibition of transformation.

[0049] To design functionally related and functionally variant JIP-1polypeptides, it is useful to distinguish between conserved positionsand variable positions. To preserve JIP-1 function, it is preferablethat conserved residues are not altered. Moreover, alteration ofnon-conserved residues are preferably conservative alterations, e.g., abasic amino acid is replaced by a different basic amino acid. To producealtered function variants, it is preferable to make non-conservativechanges at variable and/or conserved positions. Deletions at conservedand variable positions can also be used to create altered functionvariants. Conserved amino acids in JIP-1 include, but are not limitedto, Lys-155, Thr-159, Leu-160, Asn-161, and Leu-162.

[0050] Preferred JIP-1 polypeptides are those that bind JNK and inhibittransformation or apoptosis. These JIP-1 polypeptides have 20%, 40%,50%, 75%, 80%, 90%, or even greater than 100% of the activity of thefull-length JIP-1 described herein. Such comparisons are generally basedon equal concentrations of the molecules being compared. The comparisoncan also be based on the amount of protein or polypeptide required toreach 50% of the maximal stimulation obtainable.

[0051] Polypeptides corresponding to one or more domains of JIP-1, e.g.,the JNK Binding Domain (JBD), are also within the scope of theinvention. Preferred polypeptides are those which are soluble undernormal physiological conditions. Also within the invention are fusionproteins in which a portion (e.g., one or more domains) of JIP-1 isfused to an unrelated protein or polypeptide (i.e., a fusion partner) tocreate a fusion protein. The fusion partner can be a moiety selected tofacilitate purification, detection, or solubilization, or to providesome other function. Fusion proteins are generally produced byexpressing a hybrid gene in which a nucleotide sequence encoding all ora portion of JIP-1 is joined in-frame to a nucleotide sequence encodingthe fusion partner. Fusion partners include, but are not limited to, theconstant region of an immunoglobulin (IgFc). A fusion protein in which aJIP-1 polypeptide is fused to IgFc can be more stable and have a longerhalf-life in the body than the JIP-1 polypeptide on its own.

[0052] Also within the scope of the invention are various soluble formsof JIP-1, including JIP-1 expressed on its own or fused to asolubilization partner, e.g., an immunoglobulin.

[0053] In general, JIP-1 polypeptides can be produced by transformation(transfection, transduction, or infection) of a host cell with all orpart of a JIP-1-encoding DNA fragment (e.g., the cDNA described herein)in a suitable expression vehicle. Suitable expression vehicles include:plasmids, viral particles, and phage. For insect cells, baculovirusexpression vectors are suitable. The entire expression vehicle, or apart thereof, can be integrated into the host cell genome. In somecircumstances, it is desirable to employ an inducible expression vector,e.g., the LACSWITCH™ Inducible Expression System (Stratagene; LaJolla,Calif.).

[0054] Any of a wide variety of expression systems can be used toprovide the recombinant proteins. The precise host cell used is notcritical to the invention. The JIP-1 protein can be produced in aprokaryotic host (e.g., E. coli or B. subtilis) or in a eukaryotic host,e.g., yeast, such as Saccharomyces or Pichia; mammalian cells, such asCOS, NIH 3T3, CHO, BHK, 293, or HeLa cells; or insect cells.

[0055] JIP-1 polypeptides can also be produced by plant cells. Viralexpression vectors (e.g., cauliflower mosaic virus and tobacco mosaicvirus) and plasmid expression vectors (e.g., Ti plasmid) are suitablefor use in plant cells. Plant cells are available from a wide range ofsources, e.g., the American Type Culture Collection, Rockland, Md. Seealso, e.g., Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, 1995. The methods of transformation ortransfection and the choice of expression vehicle will depend on thehost system selected. Transformation and transfection methods aredescribed in, e.g., Ausubel et al., supra; expression vehicles may bechosen from those described in, e.g., Cloning Vectors: A LaboratoryManual, P. H. Pouwels et al., 1985, Supp. 1987.

[0056] The host cells harboring the expression vehicle can be culturedin conventional nutrient media adapted as needed for activation of achosen gene, repression of a chosen gene, selection of transformants, oramplification of a chosen gene.

[0057] A suitable expression system is the mouse 3T3 fibroblast hostcell transfected with a pMAMneo expression vector (Clontech, Palo Alto,Calif.). pMAMneo provides an RSV-LTR enhancer linked to adexamethasone-inducible MMTV-LTR promotor, an SV40 origin of replicationwhich allows replication in mammalian systems, a selectable neomycingene, and SV40 splicing and polyadenylation sites. DNA encoding a JIP-1in protein would be inserted into the pMAMneo vector in an orientationdesigned to allow expression. The recombinant JIP-1 protein would beisolated as described below. Other preferable host cells that can beused in conjunction with the pMAMneo expression vehicle include COScells and CHO cells (ATCC Accession Nos. CRL 1650 and CCL 61,respectively).

[0058] JIP-1 polypeptides can be expressed as fusion proteins. Forexample, the expression vector pUR278 can be used to create lacZ fusionproteins. See Ruther et al., EMBO J. 2:1791 (1983). The pGEX vectors canbe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can be easily purified from cell lysates by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

[0059] In an insect cell expression system, Autographa californicanuclear polyhidrosis virus (AcNPV), which grows in Spodoptera frugiperdacells, is used as a vector to express foreign genes. A JIP-1 codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of aAcNPV promoter, e.g., the polyhedrin promoter. Successful insertion of agene encoding a JIP-1 polypeptide or protein will result in inactivationof the polyhedrin gene and production of non-occluded recombinant virus(i.e.,, virus lacking the proteinaceous coat encoded by the polyhedringene). Spodoptera frugiperda cells are then infected with these viruses,and the inserted gene is expressed. See, e.g., Smith et al., J. Virol.46:584 (1983); Smith, U.S. Pat. No. 4,215,051.

[0060] In mammalian host cells, a number of viral-based expressionsystems can be utilized. In cases where an adenovirus is used as anexpression vector, the JIP-1 nucleic acid sequence can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence, to form a chimeric gene. Thischimeric gene can then be inserted into the adenovirus genome by invitro or in vivo recombination. Insertion into a non-essential region ofthe viral genome (e.g., the E1 or E3 gene) will result in a recombinantvirus that is viable and capable of expressing a JIP-1 gene product ininfected hosts. See, e.g., Logan, Proc. Natl. Acad. Sci. USA, 81:3655(1984).

[0061] Specific initiation signals may be required for efficienttranslation of inserted nucleic acid sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire native JIP-1 gene or cDNA, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional translational control signals may be needed. In othercases, exogenous translational control signals, including, perhaps, theATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. Exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements. Bittner et al., Methods in Enzymol., 153:516 (1987).

[0062] In addition, a host cell may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in a desired fashion. Such modifications (e.g., glycosylation)and processing (e.g., cleavage) of protein products may be important forthe function of the protein. Different host cells have characteristicand specific mechanisms for post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the foreign protein expressed. To this end, eukaryotichost cells that possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38 cells.

[0063] Alternatively, a JIP-1 protein can be produced by astably-transfected mammalian cell line. A number of vectors suitable forstable transfection of mammalian cells are available to the public. See,e.g., Pouwels et al., supra. Methods for constructing such cell linesare also publicly available. See, e.g., Ausubel et al., supra. JIP-1cDNA can be cloned into an expression vector that includes thedihydrofolate reductase (DHFR) gene. Methotrexate (0.01-300 μM) ispresent in the culture medium to select for cells which have integratedthe plasmid and, therefore, the JIP-1 cDNA. See Ausubel et al., supra.This dominant selection can be accomplished in most cell types.

[0064] Recombinant protein expression can be increased by DHFR-mediatedamplification of the transfected gene. Methods for selecting cell linesbearing gene amplifications are described in Ausubel et al., supra. Suchmethods generally involve extended culture in medium containinggradually increasing levels of methotrexate. DHFR-containing expressionvectors commonly used for this purpose include pCVSEII-DHFR andpAdD26SV(A). See Ausubel et al., supra. Any of the host cells describedabove or, preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR⁻ 0cells, ATCC Accession No. CRL 9096) are among the host cells preferredfor DHFR selection of a stably-transfected cell line or DHFR-mediatedgene amplification.

[0065] A number of other selection systems can be used, including butnot limited to the herpes simplex virus thymidine kinase,hypoxanthine-guanine phosphoribosyl-transferase, and adeninephosphoribosyltransferase genes can be employed in tk, hgprt, or aprtcells, respectively. In addition, gpt, which confers resistance tomycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci. USA, 78:2072(1981); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981)); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene, 30:147 (1981)),can be used.

[0066] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described in Janknecht et al., Proc. Natl. Acad.Sci. USA, 88:8972 (1981), allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines. In thissystem, the gene of interest is subcloned into a vaccinia recombinationplasmid such that the gene's open reading frame is translationally fusedto an amino-terminal tag consisting of six histidine residues. Extractsfrom cells infected with recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose columns, and histidine-tagged proteins areselectively eluted with imidazole-containing buffers.

[0067] Alternatively, JIP-1 or a portion thereof can be fused to animmunoglobulin Fc domain. Such a fusion protein can be readily purifiedusing a protein A column. Moreover, such fusion proteins permit theproduction of a dimeric form of a JIP-1 polypeptide having increasedstability in vivo.

[0068] After the recombinant JIP-1 protein is expressed, it is isolated.Secreted forms can be isolated from the culture media, whilenon-secreted forms must be isolated from the host cells. Proteins can beisolated by affinity chromatography. An anti-JIP-1 antibody (e.g.,produced as described herein) is attached to a column and used toisolate the JIP-1 protein. Lysis and fractionation of JIP-1protein-harboring cells prior to affinity chromatography can beperformed by standard methods. See, e.g., Ausubel et al., supra.Alternatively, a JIP-1 fusion protein, for example, a JIP-1-maltosebinding protein, a JIP-1-β-galactosidase, or a JIP-1-trpE fusionprotein, can be constructed and used for JIP-1 protein isolation. See,e.g., Ausubel et al., supra; New England Biolabs, Beverly, Mass.

[0069] Once isolated, the recombinant protein can, if desired, befurther purified, e.g., by high performance liquid chromatography usingstandard techniques. See, e.g., Fisher, Laboratory Techniques InBiochemistry And Molecular Biology, Work et al., eds., Elsevier (1980).

[0070] Polypeptides of the invention, particularly short JIP-1fragments, can also be produced by chemical synthesis, e.g., by themethods described in Solid Phase Peptide Synthesis, 2nd ed., The PierceChemical Co., Rockford, Ill., 1984.

[0071] The invention also features proteins which interact with JIP-1and which are involved in the function of JIP-1. Also included in theinvention are the genes encoding these interacting proteins. Interactingproteins can be identified using methods known to those skilled in theart. One method suitable method is the “two-hybrid system” which detectsprotein interactions in vivo. See, e.g., Chien et al., Proc. Natl. Acad.Sci. USA, 88:9578 (1991). A kit for practicing this method is availablefrom Clontech (Palo Alto, Calif.).

[0072] JIP-1 Nucleic Acids

[0073] The JIP-1 cDNA sequences described herein, and related familymembers of the JIP-1 gene present in mouse, human, or other species canbe identified and readily isolated without undue experimentation by wellknown molecular biological techniques given the specific sequencesdescribed herein. Further, genes may exist at other loci that encodeproteins having extensive homology to JIP-1 polypeptides or one or moredomains of JIP-1 polypeptides. These genes can be identified by knowntechniques using the sequences disclosed herein. For example,hybridization of JIP-1 probes to homologous nucleic acids is performedunder stringent conditions. Alternatively, a labeled fragment can beused to screen a genomic library derived from the organism of interest,again, using appropriately stringent conditions. Such stringentconditions are well known, and will vary predictably depending on thespecific organisms from which the library and the labeled sequences arederived.

[0074] Nucleic acid duplex or hybrid stability is expressed as themelting temperature, or T_(m), which is the temperature at which a probedissociates from a target DNA. This melting temperature is used todefine the required stringency conditions. If sequences are to beidentified that are related and substantially identical to the probe,rather than identical, then it is useful to first establish the lowesttemperature at which only homologous hybridization occurs with aparticular concentration of SSC or SSPE. It is then assumed that 1%mismatching results in a 1° C. decrease in the T_(m), and thetemperature of the final wash is reduced accordingly (for example, ifsequences with ≧95% identity with the probe are sought, the final washtemperature is decreased by 5° C.). Note that this assumption is veryapproximate, and the actual change in T_(m) can be between 0.50° and1.5° C. per 1% mismatch.

[0075] As used herein, stringent conditions include hybridization at 68°C. in 5×SSC/5×Denhardt's solution/1.0% SDS, or in 0.5 M NaHPO₄ (pH7.2)/1 mM EDTA/7% SDS, or in 50% formamide/0.25 M NaHPO₄ (pH 7.2)/0.25 MNaCl/1 mM EDTA/7% SDS; and washing in 0.2×SSC/0.1% SDS at roomtemperature or at 42° C., or in 0.1×SSC/0.1% SDS at 68° C., or in 40 mMNaHPO₄ (pH 7.2)/1 mM EDTA/5% SDS at 50° C., or in 40 mM NaHPO₄ (pH 7.2)1 mM EDTA/1% SDS at 50° C. Moderately stringent conditions includewashing in 3×SSC at 42° C. The parameters of salt concentration andtemperature can be varied to achieve the desired level of identitybetween the probe and the target nucleic acid. For guidance regardingsuch conditions see, e.g., Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.;and Ausubel et al., supra, at Unit 2.10.

[0076] Upon detection of JIP-1 transcript in human cell lines byNorthern blot analysis, cDNA libraries can be constructed from RNAisolated from these cell lines utilizing standard techniques. The humancDNA library can then be screened with a JIP-1 probe to isolate a humanJIP-1 cDNA.

[0077] Alternatively, a human genomic DNA library can be screened usingJIP-1 probes. Hybridizing clones can be sequenced and the intron andexon structure of the human JIP-1 gene can be elucidated. Once a genomicsequence is obtained, oligonucleotide primers can be designed based onthe sequence for use in Reverse Transcriptase-coupled PCR, e.g., toisolate human JIP-1 cDNA. An example of a suitable probe for screening ahuman genomic library is the coding region (nucleotides 180 to 2159) ofthe mouse JIP-1 cDNA (SEQ ID NO:2).

[0078] Further, a previously unknown gene sequence can be isolated byperforming PCR using two degenerate oligonucleotide primer poolsdesigned on the basis of nucleotide sequences within the JIP-1 cDNAdefined herein. Degenerate PCR primers that can be used include:5′GARGARTTYGARGAYGARGA 3′ (sense; SEQ ID NO:25); 5′GGNAARAARCAYAGNTGGCA3′ (sense; SEQ ID NO:26); 5′CATRTTWTANGCYTCWTACCA 3′ (antisense; SEQ IDNO:27); and 5′AAYTGYTTKTARAAYTGYTGRAA 3′ (antisense; SEQ ID NO:28),where W is A or T, K is G or T, R is A or G, Y is C or T, and N is A, C,G or T. The template for the reaction can be cDNA obtained by reversetranscription of mRNA prepared from human or non-human cell lines ortissue known to express, or suspected of expressing, JIP-1. The PCRproduct can be subcloned and sequenced to insure that the amplifiedsequences represent the sequences of JIP-1 or JIP-1-like gene nucleicacid sequences.

[0079] The PCR fragment can then be used to isolate a full length cDNAclone by a variety of methods. For example, the amplified fragment canbe labeled and used to screen a cDNA library in bacteriophage.Alternatively, the labeled fragment can be used to screen a genomiclibrary.

[0080] PCR technology also can be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate tissue or cell line. A reversetranscription reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid can then be “tailed” with guanines using a standardterminal transferase reaction. After digestion with RNAase H, and secondstrand synthesis can be primed with a poly-C primer. Thus, cDNAsequences upstream of the amplified fragment can easily be isolated. Fora review of useful cloning strategies, see e.g., Sambrook et al., supra;Ausubel et al., supra.

[0081] Mutant cDNAs can also be isolated using PCR techniques. The firstcDNA strand can be synthesized by hybridizing an oligo-dToligonucleotide to mRNA isolated from tissue known to be or suspected ofbeing expressed in an individual putatively carrying the mutant allele,and by extending the new strand with reverse transcriptase. The secondstrand of the cDNA can then be synthesized using an oligonucleotide thathybridizes specifically to the 5′-end of the normal gene. Using thesetwo oligonucleotides as primers, the product is amplified via PCR,cloned into a suitable vector, and subjected to DNA sequence analysis bymethods well known in the art. By comparing the DNA sequence of themutant gene to that of the normal gene, the mutation(s) responsible forthe loss or alteration of function of the mutant gene product can beascertained.

[0082] Alternatively, a genomic or cDNA library can be constructed andscreened using DNA or RNA, respectively, from a tissue known to express,or suspected of expressing, the gene of interest in an individualsuspected of carrying or known to carry the mutant allele. The normalgene or any suitable fragment thereof can then be labeled and used as aprobe to identify the corresponding mutant allele in the library. Theclone containing the mutant gene can then be purified through methodsroutinely practiced in the art, and subjected to sequence analysis usingstandard techniques as described herein.

[0083] Additionally, an expression library can be constructed using DNAisolated from or cDNA synthesized from a tissue known to express orsuspected of expressing the gene of interest in an individual suspectedof carrying or known to carry the mutant allele. In this manner, geneproducts made by this tissue can be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against the normal gene product, as described herein. Forscreening techniques, see, for example, Harlow et al., eds., Antibodies:A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1988).

[0084] In cases where the mutation results in an expressed gene productwith altered function (e.g., as a result of a missense mutation), apolyclonal set of antibodies is likely to cross-react with the mutantgene product. Library clones, detected via their reaction with suchlabeled antibodies can be purified and subjected to sequence analysis asdescribed herein.

[0085] JIP-1 Peptide Mimetics

[0086] JIP-1 peptide mimetics can be constructed by structure-based drugdesign through replacement of amino acids by organic moieties. See,e.g., Hughes, Philos. Trans. R. Soc. Lond., 290:387-394 (1980); Hodgson,Biotechnol., 9:19-21 (1991); Suckling, Sci. Prog., 75:323-359 (1991).The use of peptide mimetics can be enhanced through the use ofcombinatorial chemistry to create drug libraries. The design of peptidemimetics can be aided by identifying amino acid mutations that increaseor decrease JIP-1 binding to JNK. Approaches that can be used includethe yeast two hybrid method (see, e.g., Chien et al., Proc. Natl. Acad.Sci. USA, 88:9578 (1991); kit from Clontech, Palo Alto, Calif.), and thephage display method. The two hybrid method detects protein-proteininteractions in yeast. Fields et al., Nature, 340:245-246 (1989). Thephage display method detects the interaction between an immobilizedprotein and a protein that is expressed on the surface of phages (e.g.lambda and M13). Amberg, et al., Strategies, 6:2-4 (1993); Hogrefe etal., Gene, 128:119-126 (1993). These methods allow positive and negativeselection for protein-protein interactions and the identification of thesequences that determine these interations.

[0087] Transgenic Animals

[0088] JIP-1 polypeptides can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates,e.g., baboons, monkeys, and chimpanzees, can be used to generateJIP-1-expressing transgenic animals.

[0089] Various techniques known in the art can be used to introduce aJIP-1 transgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to, pronuclearmicroinjection (U.S. Pat. No. 4,873,191); retrovirus mediated genetransfer into germ lines (van der Putten et al., Proc. Natl. Acad. Sci.USA, 82:6148 (1985)); gene targeting into embryonic stem cells (Thompsonet al., Cell, 56:313 (1989)); and electroporation of embryos (Lo, Mol.Cell. Biol, 3:1803 (1983)).

[0090] The present invention provides for transgenic animals that carrythe JIP-1 transgene in all their cells, as well as animals that carrythe transgene in some, but not all of their cells, i.e., mosaic animals.The transgene can be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems. The transgene canalso be selectively introduced into and activated in a particular celltype. Lasko et al., Proc. Natl. Acad. Sci. USA, 89:6232 (1992). Theregulatory sequences required for such a cell-type specific activationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

[0091] When it is desired that the JIP-1 transgene be integrated intothe chromosomal site of the endocenous JIP-1 gene, gene targeting ispreferred. Vectors containing some nucleotide sequences homologous to anendogenous JIP-1 gene are designed for the purpose of integrating viahomologous recombination into the endogenous gene and disrupting itsfunction. The transgene also can be selectively introduced into aparticular cell type, thus inactivating the endogenous JIP-1 gene inonly that cell type. See Gu et al., Science, 265:103 (1984). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

[0092] Once transgenic animals have been generated, the expression ofthe recombinant JIP-1 gene can be assayed utilizing standard techniques.Initial screening can be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals can also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of tissues expressing JIP-1 can also beevaluated immunocytochemically using antibodies specific for the JIP-1transgene product.

[0093] Anti-JIP-1 Antibodies

[0094] Since JIP-1 is an inhibitor of JNK, inhibition of JIP-1 increasesJNK activity. Increased JNK expression results in increased apoptosis,e.g., in neurons. Induction of apoptosis would be desirable in braintumors, for example. Therefore, antibodies specific for JIP-1 can beused to inhibit JIP-1 expression.

[0095] Human JIP-1 proteins and polypeptides (or immunogenic fragmentsor analogs) can be used to raise antibodies; such polypeptides can beproduced by recombinant or peptide synthetic techniques. See, e.g.,Solid Phase Peptide Synthesis, supra; Ausubel et al., supra. In general,the peptides can be coupled to a carrier protein, such as KLH, mixedwith an adjuvant, and injected into a host mammal. Antibodies can bepurified by peptide antigen affinity chromatography.

[0096] In particular, various host animals can be immunized by injectionwith a JIP-1 protein or polypeptide. Host animals include rabbits, mice,guinea pigs, and rats. Various adjuvants can be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete or incomplete); mineral gels, such asaluminum hydroxide; surface active substances such as lysolecithin;pluronic polyols; polyanions; peptides; oil emulsions; keyhole limpethemocyanin; dinitrophenol; and potentially useful human adjuvants suchas BCG (bacillus Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

[0097] The invention includes monoclonal antibodies, polyclonalantibodies, humanized or chimeric antibodies, single chain antibodies,Fab fragments, F(ab′)₂ fragments, and molecules produced using a Fabexpression library.

[0098] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be prepared using the JIP-1polypeptides described above and standard hybridoma technology. See,e.g., Kohler e: al., Nature, 256:495 (1975); Kohler et al., Eur. J.Immunol., 6:511 (1976); Kohler et al., Eur. J. Immunol., 6:292 (1976)Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas,Elsevier, N.Y. (1981); Ausubel et al., supra.

[0099] In particular, monoclonal antibodies can be obtained by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture. Such methods include those describedin Kohler et al., Nature 256:495 (1975), and U.S. Pat. No. 4,376,110.Other methods of producing monoclonal antibodies include the humanB-cell hybridoma technique (Kozbor et al., Immunology Today 4:72 (1983);Cole et al., Proc. Natl. Acad. Sci. USA 80:2026 (1983), and theEBV-hybridoma technique (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96 (1983)). Such antibodies can beof any immunoglobulin class, e.g., IgG, IgM, IgE, IgA, IgD, and anysubclass thereof. Hybridomas producing the monoclonal antibodies of theinvention may be cultivated in vitro or in vivo. The ability to producehigh titers of monoclonal antibodies in vivo makes this the presentlypreferred method of production.

[0100] Polyclonal or monoclonal antibodies are tested for specific JIP-1recognition by Western blot or immunoprecipitation analysis by standardmethods. See, e.g., Ausubel et al., supra. Antibodies that specificallyrecognize and bind to JIP-1 are useful in the invention. Theseantibodies can be used in immunoassays to monitor the level of JIP-1produced by a mammal, e.g., to determine the amount or subcellularlocation of JIP-1).

[0101] Preferably, antibodies of the invention are produced using JIP-1polypeptides that correspond to regions of the JIP-1 protein that lieoutside highly conserved regions and appear likely to be antigenic, bycriteria such as high frequency of charged residues. Such fragments canbe generated by standard PCR techniques and then cloned into the pGEXexpression vector. Ausubel et al., supra. Fusion proteins are expressedin E. coli and purified using a glutathione agarose affinity matrix asdescribed in Ausubel, et al., supra.

[0102] In some cases it may be desirable to minimize the potentialproblems of low affinity or specificity of antisera. In suchcircumstances, two or three fusion proteins can be generated for eachprotein, and each fusion protein can be injected into at least tworabbits. Antisera can be raised by injections in a series, preferablyincluding at least three booster injections.

[0103] Antisera is also checked for its ability to immunoprecipitaterecombinant JIP-1 proteins or control proteins, such as glucocorticoidreceptor, CAT, or luciferase.

[0104] The antibodies can be used to detect JIP-1 in a biological sampleas part of a diagnostic assay. Antibodies also can be used in ascreening assay to measure the effect of a candidate compound onexpression or localization of JIP-1. Additionally, such antibodies canbe used in conjunction with the gene therapy techniques, e.g., toevaluate the normal and/or engineered JIP-1-expressing cells prior totheir introduction into the patient. Such antibodies additionally can beused in a method for inhibiting abnormal JIP-1 activity. Such abnormalactivity includes altered apoptosis and proliferation, resulting inneurodegenerative diseases, autoimmune disease, cancers such asleukemias. Other abnormal JIP-1 activity includes damage caused byischemia/reperfusion, especially in heart disease, kidney damage, andstroke.

[0105] For theraputic uses, murine or other monoclonal antibodies shouldbe altered to make them less immunogenic when administered to humanpatients. For example, techniques have been developed for the productionof “chimeric antibodies.” See Morrison et al., Proc. Natl. Acad. Sci.,81:6851 (1984); Neuberger et al., Nature, 312:604 (1984); Takeda et al.,Nature, 314:452 (1984). These techniques involve splicing the genes froma mouse antibody molecule of appropriate antigen specificity togetherwith genes from a human antibody of appropriate biological activity.Such chimeric antibodies have, e.g., a variable region derived from amurine antibody and a constant region derived from a human antibody.

[0106] Alternatively, single chain antibodies specific for JIP-1polypeptides can be produced using known techniques. See, e.g., U.S.Pat. Nos. 4,946,778 and 4,704,692. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

[0107] Antibody fragments that recognize and bind to specific epitopesof JIP-1 can also be generated by known techniques. Such fragmentsinclude, but are not limited to, F(ab′)₂ fragments, produced by pepsindigestion of antibody molecules, and Fab fragments, generated byreduction of the disulfide bonds of F(ab′)₂ fragments. Alternatively,Fab expression libraries can be constructed to allow rapid and easyidentification of monoclonal Fab fragments with a desired specificity.See, e.g., Huse et al., Science 246:1275 (1989).

[0108] Antibodies to JIP-1 can be used to generate anti-idiotypicantibodies that resemble a portion of JIP-1, using techniques well knownto those skilled in the art. See, e.g., Greenspan et al., FASEB J. 7:437(1993); Nisonoff, J. Immunol. 147:2429 (1991). For example, antibodiesthat bind to JIP-1 and competitively inhibit binding of other ligandscan be used to generate anti-idiotypic antibodies resembling a ligandbinding domain of JIP-1. These anti-idiotypic antibodies can bind andneutralize JIP-1 ligands. JIP-1 ligands include proline-rich regions ofproteins, since JIP-1 has an SH3 domain that binds to these regions.There are ten isoforms of JNK. Gupta et al., EMBO J. 15:2760-2770(1996). Each of these JNK isoforms is a JIP-1 ligand. Other kinases,which may be related to JNK, may also interact with JIP-1. Neutralizinganti-idiotypic antibodies or Fab fragments of anti-idiotypic antibodiescan be used in therapeutic regimens.

[0109] Antisense Nucleic Acids

[0110] In alternate embodiments, therapies can be designed to reduce thelevel of endogenous JIP-1 gene expression, e.g., using antisense orribozyme approaches to inhibit or prevent translation of JIP-1 mRNAtranscripts; triple helix approaches to inhibit transcription of theJIP-1 gene; or targeted homologous recombination to inactivate or “knockout” the JIP-1 gene or its endogenous promoter. Delivery techniques canbe designed to allow theraputic compositions to cross the blood-brainbarrier (see, e.g., PCT WO89/10134).

[0111] Antisense approaches involve the design of oligonucleotides(either DNA or RNA) that are complementary to JIP-1 mRNA and inhibitexpression of JIP-1 protein. Absolute complementarity of the antisenseoligonucleotide to JIP-1, although preferred, is not required. Asequence “complementary” to a portion of an RNA, as referred to herein,means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may be tested, or triplex formation may be assayed. The ability tohybridize will depend on both the degree of complementarily and thelength of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

[0112] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation condon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well (Wagner, Nature, 372:333, 1994). Thus,oligonucleotides complementary to either the 5′- or 3′-non-translated,non-coding regions of the JIP-1 gene can be used in an antisenseapproach to inhibit translation of endogenous JIP-1 mRNA.Oligonucleotides complementary to the 5′ region overlapping theinitiation codon can be used for this purpose. Antisenseoligonucleotides that are complementary to the coding region can also beused. In designing suitable oligonucleotides, target regions aregenerally those lacking predicted secondary structure.

[0113] Antisense oligonucleotides complementary to mRNA coding regionscan also be used in accordance with the invention. Whether designed tohybridize to the 5′, 3′, or coding region of JIP-1 mRNA, antisensenucleic acids should be at least six nucleotides in length, and arepreferably oligonucleotides ranging from 6 to about 50 nucleotides inlength. In specific aspects the oligonucleotide is at least 10nucleotides, at least 17 nucleotides, at least 25 nucleotides or atleast 50 nucleotides in length.

[0114] It is preferred that in vitro studies are first performed toquantitate the ability of the antisense oligonucleotide to inhibit geneexpression. These studies preferably utilize controls that distinguishbetween antisense inhibition and nonspecific biological effects ofoligonucleotides. It is also preferred that these studies compare levelsof the target RNA or protein with that of an internal control RNA orprotein. Additionally, it is envisioned that results obtained using theantisense oligonucleotide are compared with those obtained using acontrol oligonucleotide. It is preferred that the controloligonucleotide is of approximately the same length as the testoligonucleotide and that the nucleotide sequence of the oligonucleotidediffers from the antisense sequence no more than is necessary to preventspecific hybridization to the target sequence.

[0115] The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. Stability of the oligonucleotide can be improved bymodification of the phosphodiester moieties by replacement withphosphorothioate, —CH2—, or —NH— groups. The oligonucleotides can alsobe stabilized by blocking the 5′ and 3′ termini with phosphorothioate,—CH2— or —NH— groups. Each of these modifications will lead to increasedstability (e.g. by increasing modifications will lead to increasedstability (e.g. by increasing resistance to nucleases) and thereforeincreased hybridization. The oligonucleotide can be modified at the basemoiety, sugar moiety, or phosphate backbone. The oligonucleotide mayinclude other appended groups such as peptides (e.g., for targeting hostcell receptors in vivo), or agents facilitating transport across thecell membrane (as described, e.g., in Letsinger et al., Proc. Natl.Acad. Sci. USA, 86:6553 (1989); Lemaitre et al., Proc. Natl. Acad. Sci.USA, 84:648 (1987)); PCT Publication No. WO 88/09810) or the blood-brainbarrier (see, e.g., PCT Publication No. WO 89/10134), orhybridization-triggered cleavage agents (see, e.g., Krol et al.,BioTechniques 6:958 (1988)), or intercalating agents (see e.g., Zon,Pharm. Res. 5:539 (1988)). To this end, the oligonucleotide can beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, or hybridization-triggeredcleavage agent.

[0116] The antisense oligonucleotide can include at least one modifiedbase moiety which is selected from the group including, but not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethyl-aminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylcytosine, N6-adenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-theouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 2-(3-amino-3-N-2-carboxypropl) uracil, (acp3)w,and 2,6-diaminopurine.

[0117] The antisense oligonucleotide can also include at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0118] In yet another embodiment, the antisense oligonucleotide includesat least one modified phosphate backbone selected from the groupconsisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal, or ananalog of any of these backbones.

[0119] In yet another embodiment, the antisense oligonucleotide is aná-anomeric oligonucleotide. An á-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual á-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids. Res., 15:6625 (1987)). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131(1987)), or a chimeric RNA-DNA analog (Inoue et al., FEBS Lett., 215:327(1987)).

[0120] Antisense oligonucleotides of the invention can be synthesized bystandard methods known in the art, e.g., by use of an automated DNAsynthesizer (commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesizedby the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. USA,85:7448 (1988)).

[0121] The antisense molecules should be delivered to cells that expressJIP-1 in vivo. A number of methods have been developed for deliveringantisense DNA or RNA to cells. For example, antisense molecules can beinjected directly into specific tissue sites. Modified antisensemolecules, designed to target the desired cells (e.g., antisense linkedto peptides or antibodies that specifically bind receptors or antigensexpressed on the target cell surface) can be administered systemically.

[0122] One approach to achieving intracellular concentrations of theantisense molecule sufficient to suppress translation of endogenousmRNAs is to use a recombinant DNA construct in which the antisenseoligonucleotide is placed under the control of a strong pol III or polII promoter. The use of such a construct to transfect target cells in apatient will result in the transcription of sufficient amounts of singlestranded RNAs that will form complementary base pairs with theeldogenous JIP-1 transcripts, and thereby prevent translation of theJIP-1 mRNA. For example, a vector can be introduced in vivo such that itis taken up by a cell and directs the transcription of an antisense RNA.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA.

[0123] Such vectors can be constructed by recombinant DNA technologymethods standard in the art. Vectors can be plasmid, viral, or othersknown in the art, used,for replication and expression in mammaliancells. Expression of the sequence encoding the antisense RNA can be byany promoter known in the art to act in mammalian, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude, but are not limited to: the SV40 early promoter region(Bernoist et al., Nature, 290:304 (1981)); the promoter contained in the3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell,22:787-797 (1988)); the herpes thymidine kinase promoter (Wagner et al.,Proc. Natl. Acad. Sci USA, 78:1441, 1981); or the regulatory sequencesof the metallothionein gene (Brinster et al., Nature, 296:39 (1988)).

[0124] Any type of plasmid, cosmid, YAC, or viral vector can be used toprepare the recominant DNA construct which can be introduced directlyinto specific tissue sites. Alternatively, viral vectors can be usedthat selectively infect the desired tissue (e.g., for brain, herpesvirusvectors may be used), in which case administration can be accomplishedby another route (e.g., systemically).

[0125] Ribozymes

[0126] Ribozyme molecules designed to catalytically cleave JIP-1 mRNAtranscripts also can be used to prevent translation of JIP-1 mRNA andexpression of JIP-1 protein (see, e.g., PCT Publication WO 90/11364;Saraver et al., Science, 247:122 (1990)). While various ribozymes thatcleave mRNA at site-specific recognition sequences can be used todestroy JIP-1 mRNAs, the use of hammerhead ribozymes is preferred.Hammerhead ribozymes cleave mRNAs at locations dictated by flankingregions that form complementary base pairs with the target mRNA. Thesole requirement is that the target mRNA contain the following sequenceof two bases: 5′-UG-3′. The construction and production of hammerheadribozymes is well known in the art (Hasseloff et al., Nature, 334;585,1988).

[0127] The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”), such as the onethat occurs naturally in Tetrahymena Thermophila (known as the IVS orL-19 IVS RNA), which have been extensively characterized by Cech and hiscollaborators. See, e.g., Zaug et al., Science, 224:574 (1984); Zaug etal., Science, 231:470 (1986); Zaug et al., Nature, 324:429 (1986); PCTApplication No. WO 88/04300; and Been et al., Cell, 47:207 (1986). TheCech-type ribozymes have an eight base-pair sequence that hybridizes toa target RNA sequence, whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes that targeteight base-pair active site sequences present in JIP-1. In general,target sites for ribozymes are regions that are predicted to lackappreciable secondary structure. Target sites for ribozymes include:

[0128] 5′GGUAUCGAUAAGCUUGAUAUCGCUGUCCGGAGC 3′ (SEQ ID NO:29) and

[0129] 5′AGAGGCACUGUCCCAUCCUGGGCCUGUUUCAUG 3′ (SEQ ID NO:30).

[0130] As in the antisense approach, the ribozymes can be composed ofmodified oligonucleotides (e.g., for improved stability, targeting,etc.), and should be delivered to cells which express JIP-1 in vivo. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the rihozyme to destroy endogenous JIP-1 messages andinhibit translation. Because ribozymes, unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency. Destruction of JIP-1 mRNA would be advantageous inincreasing apoptosis in tumors; in altering the cell damage occurringduring ischemic reperfusion in cardiovascular disease, kidney disease,and stroke; and altering cell damage occurring in the liver in responseto metabolic oxidative stress.

[0131] Other Methods for Reducina JIP-1 Expression

[0132] Endogenous JIP-1 gene expression can also be reduced byinactivating or “knocking out” the JIP-1 gene or its promoter usingtargeted homologous recomination (see, e.g., U.S. Pat. No. 5,464,764).For example, a mutant, non-functional JIP-1 (or a completely unrelatedDNA sequence) flanked by DNA homologous to the endogenous JIP-1 gene(either the coding regions or regulatory regions of the JIP-1 gene) canbe used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express JIP-1 in vivo.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the JIP-1 gene. Such approaches areparticularly suited for use in the agricultural field wheremodifications to ES (embryonic stem) cells can be used to generateanimal offspring with an inactive JIP-1. However, this approach can beadapted for use in humans, provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors, e.g., herpes virus vectors for delivery tobrain tissue.

[0133] Alternatively, endogenous JIP-1 gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to regulatoryregions of the JIP-1 gene (e.g., a JIP-1 promoter and/or enhancer toform triple helical structures that prevent transcription of theJIP-1gene in target cells in the body. See, e.g., Helene, AnticancerDrug Des. 6:569 (1981); Helene et al., Ann N.Y. Acad. Sci. 660:27(1992); Maher, Bioassays 14:807 (1992).

[0134] Identification of Proteins that Interact with JIP-1

[0135] The invention also features polypeptides that interact withJIP-1. Any method suitable for detecting protein-protein interactionscan be employed for identifying intracellular or extracellular proteinsthat interact with JIP-1. Among the traditional methods that can beemployed are co-immunoprecipitation, crosslinking, and co-purificationthrough gradients or chromatographic columns of cell lysates, orproteins obtained from cell lysates. Purified proteins are used toidentify proteins in the lysate that interact with JIP-1. For theseassays, the JIP-1 polypeptide can be full length JIP-1, a soluble domainof JIP-1, or some other suitable JIP-1 polypeptide.

[0136] To characterize JIP-1 interacting proteins, portions of theiramino acid sequences can be ascertained using techniques well known tothose of skill in the art, such as the Edman degradation technique. Theamino acid sequences obtained can be used to design degenerateoligonucleotide probes that can be used to screen for gene sequencesencoding the interacting protein. Screening may be accomplished, forexample, by standard hybridization or PCR techniques. Techniques for thegeneration of degenerate oligonucleotide mixtures and screening arewell-known. See Ausubel, supra; and Innis et al., eds., PCR Protocols: AGuide to Methods and Applications, Academic Press, Inc., New York(1990).

[0137] Additionally, methods can be used that result directly in theidentification of genes that encode proteins that interact with JIP-1.These methods include, for example, screening expression libraries in amanner similar to the well known technique of antibody probing of λgt11libraries using labeled JIP-1 polypeptides or a JIP-1 fusion protein,e.g., an JIP-1 polypeptide or domain fused to a marker such as anenzyme, a fluorescent dye, a luminescent protein, or to an IgFc domain.

[0138] Protein interactions can also be identified in vivo using thetwo-hybrid system. Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578(1991). A kit for practicing this method is available from Clontech(Palo Alto, Calif.). In this system, two plasmids, each encoding ahybrid protein, are constructed. One plasmid, the “bait” plasmid,includes a nucleotide sequence encoding the DNA-binding domain of atranscription activator protein fused to a nucleotide sequence encodinga protein of interest. The other plasmid includes a nucleotide sequenceencoding the transcription activator protein's activation domain and anunknown protein, which may bind the protein of interest. The plasmidsare transformed into a strain of Saccharomyces cerevisiae that containsa reporter gene (e.g., HIS or lacZ) whose regulatory region contains thetranscription activator's binding site. Neither of the hybrid proteinscan activate transcription of the reporter gene alone; the bait plasmidlacks an activation function, and the other plasmid cannot localize tothe transcription activator's binding sites. If the protein of interestand the unknown protein form a protein-protein complex and reconstitutethe proximity of the DNA binding domain and the activation domain, thiscomplex can bind to the regulatory region of the reporter gene, and thereporter gene will be expressed.

[0139] The two-hybrid system or related methodology can be used toscreen libraries for proteins that interact with the “bait” geneproduct. By way of example, JIP-1 can be used as the bait gene product.Total genomic or cDNA sequences are fused to the DNA encoding anactivation domain of a transcriptional activator protein. This libraryand a plasmid encoding a hybrid of bait JIP-1 gene product fused to theDNA-binding domain are cotransformed into a yeast reporter strain, andthe resulting transformants are screened for those that express thereporter gene. For example, a bait JIP-1 gene sequence, such as JIP-1 ora domain of JIP-1, can be cloned into a vector such that it istranslationally fused to the DNA encoding the DNA-binding domain of theGAL4 protein, to form a bait plasmid. Colonies are purified and thelibrary plasmids responsible for reporter gene expression are isolated.The library plasmids are subjected to DNA sequencing to determine thesequences of the gene encoding the JIP-1 binding proteins.

[0140] As an example, a cDNA library can be made by methods that areroutine in the art. This library can be inserted into vectors so thatthe sequences from the library are fused to nucleotide sequencesencoding the transcriptional activation domain of the GAL4 protein.These vectors can then be co-transformed, along with a bait JIP-1gene-GAL4 fusion plasmid, into a yeast strain which contains a lacZ genedriven by a promoter that contains the GAL4 activation sequence. If anyof the library vectors encode hybrid proteins that bind the hybrid JIP-1protein encoded by the bait plasmid, a functional GAL4 will be formed,and the HIS3 gene will be expressed. Colonies expressing HIS3 can thenbe purified, and used to produce and isolate the JIP-1 interactingprotein using techniques routinely practiced in the art.

[0141] Therapeutic Compositions of JIP-1 Nucleic Acids, Peptides, andPolypeptides

[0142] Therapeutic compositions of the JNK inhibitor JIP-1 can be usedto treat pathological conditions associated with apoptosis ortransformation. These compositions can include JIP-1 polypeptides orpeptides that specifically bind to and sequester JNK. Proteins can bepurified by methods known to those skilled in the art. Ausubel, supra.Peptides can be synthesized by methods that are known to those skilledin the art. See, e.g., Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963).

[0143] Peptide mimetics are also included in the therapeuticcompositions of the invention. These compounds mimic the activity of thepeptide or polypeptide, but are composed of molecules other than, or inaddition to, amino acids. The design of such mimetics is described in,e.g., Hughes, Philos. Trans. R. Soc. Lond., 290:387-394 (1980); Hodgson,Biotechnol., 9:19-21 (1991); Suckling, Sci. Prog., 75:323-359 (1991).

[0144] Therapeutic compositions of the invention also include nucleicacids encoding a JIP-1 polypeptide or peptide. These nucleic acids canbe administered in a manner allowing their uptake and expression bycells in vivo. Compositions containing nucleic acids can be prepared foradministration by methods that are routine in the art.

[0145] Therapeutic compositions of the invention can include one or morecompounds, e.g., nucleic acids, peptides, polypeptides, or peptidemimetics and a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers are biologically compatible vehicles, e.g.,physiological saline, which are suitable for administration to apatient.

[0146] Nucleic acids can be administered to the patient by standardvectors and/or gene delivery systems. Suitable gene delivery systemsinclude liposomes, receptor-mediated delivery systems, naked DNA andviral vectors such as herpes viruses, retroviruses, adenoviruses andadeno-associated viruses.

[0147] Peptides can be coupled to membrane permeable peptides in orderto facilitate their uptake by cells. This can be done by colinearsynthesis of a membrane permeable peptide with a peptide sequence ofinterest. The two peptides could also be crosslinked together. Methodsof coupling are described in, e.g., Lin et al., J. Biol. Chem.,269:12320-12324 (1996); Rojas et al., J. Biol. Chem., 271:27456-127461(1997). Peptides can also be coupled to lipids to provide membranepermeability. See, e.g., Vijayaraghavan, J. Biol. Chem., 272:4747-4752(1997). As an example, a fatty acid can be coupled to the α-NH₂ group ofthe peptide as an amide.

[0148] To enable the compositions to penetrate the blood-brain barrier,they can be delivered in encapsulated cell implants (e.g., thoseproduced by CytoTherapeutics, Inc., Providence R.I.; see Bioworld Today7:6 (Monday, Dec. 2, 1996)). Delivery of drugs to the brain can also beaccomplished using RMP-7™ technology (Alkermes, Inc., Cambridge, Mass.;see Business Wire, “Third Major Agreement for Prolease Sustained ReleaseDrug Delivery System,” Dec. 2, 1996) or implantable wafers containingthe drug (see PR Newswire, “Implantable Wafer is First Treatment toDeliver Chemotherapy Directly to Tumor Site,” Sep. 24, 1996). Thecompositions can also be administered using an implantable pump fordirect administration into intrathecal fluid (e.g., that made byMedtronic, Minneapolis, Minn.; see Genetic Engineering News,“Neurobiotechnology Companies Focus Programs on Pain andNeuroprotection,” Nov. 1, 1996).

[0149] Administration of Therapeutic Compositions

[0150] Parenteral administration, such as intravenous, subcutaneous,intramuscular, or intraperitoneal delivery routes can be used to deliverthe therapeutic compositions of the invention. Dosages for particularpatients depend upon many factors, including the patient's size, bodysurface area, age, the particular substance to be administered, time androute of administration, general health and other drugs beingadministered concurrently. The amount of therapeutic composition to beadministered to a patient can be in the range of 1 to 1000 μg/kg of bodyweight, e.g., 10 to 500 μg/kg, or 20 to 200 μg/kg of body weight. Atypical dose of peptide or nucleic acid to be administered to a patientis 100 μg per kilogram of body weight.

EXAMPLES Example 1 Identification of JIP-1

[0151] The yeast two hybrid method was used to screen a mouse embryocDNA library to identify proteins that interact with JNK. The method isdescribed in detail in Fields et al., Nature, 340:245 (1989). The yeaststrain used was L40 (MATa hisΔ200 trp1-901 leu2-3, 112 ade2 LYS::(lexAop)₄-HIS URA3:: (lexAop)₈-lacZ. Vojtek, et al., Cell, 74:205(1993). Human JNK1 fused to the LexA DNA binding domain was used as thebait. The bait plasmid, pLexA-JNK1, was constructed by blunt-endligation of a XbaI/HindIII fragment of pCMV-Flag-JNK1 (Derijard et al.,Cell, 76:1025 (1994); Sluss et al., Mol. Cell Biol., 14:8376 (1994);Gupta et al., EMBO. J., 15:2760 (1996)) into the vector pBTM116 at theSmaI and SalI sites. Yeast transformants (5.2×10⁶) were examined forgrowth on media with 25 mM 3-aminotriazole in the absence of histidine.Positive clones were confirmed by measurement of lacZ expression.

[0152] The two-hybrid screen yielded a cDNA fragment encoding a portionof a JNK binding protein. A group of 7 independent clones, correspondingto overlapping fragments of this cDNA, was identified by sequencing.Full-length cDNA clones were obtained by screening a mouse brain λZAPIIcDNA library (Stratagene Inc.) with a random-primed cDNA fragmentcorresponding to base pairs 560-1020 of the full length cDNA. IsolatedcDNA clones were sequenced using an Applied Biosystems model 373Amachine. The sequence of the 5′ GC-rich non-coding region of the largestcDNA clone was confirmed using the Maxam-Gilbert method. Maxam et al.,Proc. Natl. Acad. Sci. USA, 74:560-564 (1977). The amino acid sequenceof the encoded protein was deduced from the cDNA sequence. Single letterabbreviations for the amino acid residues are as follows: A, Ala; C,Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M,Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; andY, Tyr.

[0153] The largest cDNA clone isolated from the mouse brain library was2790 base pairs in length, and contained a predicted coding region of660 amino acids in the same reading frame as the partial clones obtainedin the two-hybrid screen. The protein encoded by this cDNA, designatedJNK interacting protein-1 (JIP-1), contains an amino terminal JNKbinding domain (defined by the overlapping 2-hybrid clones) and aputative SH3 domain in the carboxy terminus. The structure and aminoacid and nucleotide sequences of JIP-1 are shown in FIGS. 1A-1C. The JNKbinding domain (JBD; residues 127-281, SEQ ID NO:4) and the putative SH3domain (residues 491-540, SEQ ID NO:13) are indicated by boxes. Theputative SH3 domain of JIP-1 is highly related to the SH3 domainslocated in the tyrosine kinase c-fyn and the p85 subunit of PI-3′kinase.

[0154] Human homologs of the murine JIP-1 gene can be isolated using themurine JIP-1 cDNA clones or fragments of those clones as probes, bymethods that are routine in the art of molecular biology. For example, alibrary of human cDNA can be screened with a fragment of murine JIP-1cDNA, and human cDNA hybridizing to the murine JIP-1 cDNA can beisolated, cloned, sequenced and analyzed for structural and functionalsimilarity to murine JIP-1. For general methods, of isolating andcharacterizing homologous genes from libraries, see Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2d ed. 1989).

[0155] The tissue distribution of Jip-1 mRNA was examined by Northernblot analysis of poly (A) RNA isolated from different murine tissues.Northern blots were performed using 2 μg of polyA⁺ RNA isolated fromvarious murine tissues (Clontech). The blots were hybridized to a probethat was prepared by labeling JIP-1 cDNA (base pairs 515-970) with[α-³²P] dCTP (Amersham International PLC) by the random priming method(Stratagene Inc.) according to the manufacturer's instructions. Theintegrity of the mRNA samples was confirmed by hybridization to an actinprobe. The blots were washed three times with 1×SSC, 0.05% SDS, and 1 mMEDTA prior to autoradiography. The results indicate that JIP-1 isexpressed in many different tissues, including brain, heart, spleen,lung, liver, muscle, kidney and testis. Highest amounts of JIP-1 mRNAwere detected in brain, kidney, and testis.

Example 2 JIP-1 Specifically Binds JNK in vivo

[0156] To test whether JIP-1 and JNK interact in vivo,co-immunoprecipitation analysis was performed. COS-′1 cells weremock-transfected or transfected with JIP-1 and JNK1 expression vectors.Constructs expressing epitope (HA) tagged JNK1 have been describedpreviously. Dérijard et al., supra; Sluss et al., supra; Gupta et al.,supra. Mammalian JIP-1 expression vectors were constructed by subcloningthe JIP-1 cDNA into the XbaI and HindIII sites of pCMV5 and the HindIIIand EcoRI sites of pcDNA3 (Invitrogen Inc.). DNA encoding the Flagepitope, -Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-(SEQ ID NO:14) (ImmunexCorp.), was inserted into JIP-1 cDNA between the DNA encoding the firsttwo codons of JIP-1 using insertional overlapping polymerase chainreaction (PCR). Ho et al., Gene, 77:51 (1989). Bacterial JIP-1expression vectors were constructed by subcloning PCR fragments of theJIP-1 cDNA into the EcoRI and XbaI sites of pGEX-3×(Pharmacia LKBBiotechnology Inc.). GST fusion proteins were purified by affinitychromatography on GSH-agarose as described previously. Smith et al.,Gene, 67:31 (1988). Sequences of the constructs were confirmed using anApplied Biosystems model 373A machine.

[0157] For the co-immunoprecipitation experiments, transfected ormock-transfected cells were irradiated with or without UV-C (40 J/m²)and incubated for one hour. Lysates prepared from the cells wereexamined by protein immunoblot analysis using a mixture of antibodiesspecific for Flag and HA to detect Flag-JIP-1 and HA-JNK1, respectively.Cells were lysed in TLB (20 mM Tris (pH 7.5), 1% Triton X-100, 10%glycerol, 0.137 M NaCl, 25 mM sodium β-glycerophosphate, 1 mm sodiumorthovanadate, 2 mM sodium pyrophosphate, 2 mM EDTA, 10 μg/ml leupeptin,1 mM phenylmethylsulfonylfluoride). Soluble extracts were prepared bycentrifugation at 100,000×g for 30 minutes at 4° C. The extracts werepre-cleared using protein G-Sepharose (Pharmacia-LKB BiotechnologiesInc.) and incubated for one hour with monoclonal antibody to the Flagepitope (M2; IBI-Kodak) or the HA epitope (12CA5; Boehringer-Mannheim)pre-bound to protein G-Sepharose. The immunoprecipitates were washedthree times with TLB and once with 25 mM Hepes (pH 7.5), 0.2% (w/v)Triton X-100, 1 mM EDTA. Proteins were fractionated by SDS-PAGE andtransferred electrophoretically to Immobilon-P membranes (Millipore).The membranes were blocked with 10% gamma globulin-free horse serum(Gibco-BRL) and probed with the M2 monoclonal antibody, to detectFlag-JIP-1, and either the 12CA5 monoclonal antibody or a sheep anti-JNKpolyclonal antibody, to detect HA-JNK1. Immune complexes were detectedwith a second antibody coupled to horseradish peroxidase and enhancedchemiluminescence (Amersham International PLC)

[0158] JIP-1 was detected in JNK1 immunoprecipitates by proteinimmunoblot analysis, and JNK1 was detected in JIP-1 immunoprecipitates.Co-immunoprecipitation of JIP-1 with JNK2, was also observed. These dataindicate that JIP-1 specifically binds JNK in vivo. Exposure to UVradiation caused no significant change in the amount of the JNK/JIP-1complex detected by co-immunoprecipitation analysis. Control experimentsperformed to examine the specificity of the interaction of JIP-1 withJNK demonstrated that the related MAP kinases ERK2 and p38 did notco-immunoprecipitate with JIP-1. The absence of co-immunoprecipitationof JIP-1 with these MAP kinases demonstrates that JIP-1 forms a specificcomplex with JNK.

[0159] This same assay can be used to determine whether a specificpolypeptide is a JIP-1 polypeptide.

Example 3 JIP-1 Interacts Directly with JNK

[0160] To test whether JIP-1 interacts directly with JNK, in vitrobinding assays were performed. The putative JNK binding domain (JBD;amino acid residues 127-281 of JIP-1), defined by the clones obtained inthe two-hybrid screen, was expressed as a glutathione-S-transferase(GST) fusion protein. GST-fusion proteins were purified by affinitychromatography on GSH-agarose as described previously. Smith et al.,Gene, 67:31 (1988). Recombinant JNK, prepared by in vitro translation inthe presence of [³⁵S]methionine (Dérijard et al., supra; Sluss et al.,supra; Gupta et al., supra), was incubated with GST-JIP-1 immobilized onGSH-agarose. Control experiments revealed no detectable binding of JNKisoforms to GST alone. In contrast, in assays using GST-JNK fusionproteins representing ten different human JNK isoforms, each of theseproteins exhibited similar amounts of binding to JIP-1. JIP-1 thereforeinteracts with multiple JNK isoforms.

[0161] Previous studies have demonstrated that JNK binds to thetranscription factors c-Jun and ATF2, and that the binding of JNK tothese transcription factors is isoform-dependent. To test whether JIP-1binding to JNK is also isoform-dependent, a binding assay was utilized.Cell lysates (1 ml in TLE) containing JNK1, JNK2, or p38 MAP kinase,each tagged with the Flag epitope, were incubated with 5 μg GST-fusionproteins pre-bound to 10 μl glutathione-Sepharose. The GST fusionproteins used contain residues 127-202 (SEQ ID NO:15), 203-281 (SEQ IDNO:16), 164-240 (SEQ ID NO:17), or 127-281 (SEQ ID NO:4) of JIP-1. TheGST-ATF2 fusion protein contains residues 1-109 of ATF2, and the GST-Junfusion protein contains residues 1-79 of c-Jun. After incubation for onehour at 4° C. and three washes with TLB, bound proteins were detected byprotein immunoblot analysis with the M2 monoclonal antibody, which isspecific for the Flag epitope. Dérijard, et al., supra; Sluss et al.,supra; Gupta et al., supra. When ATF2 or c-jun GST fusion proteins wereused in this assay, binding to JNK1 was greater than binding to JNK2.This finding is consistent with the results of previous studies. Incontrast, when GST-JIP-1 fusion proteins containing residues 127-202 or127-281 of JIP-1 were tested in the assay, these proteins bound bothJNK1 and JNK2. The level of binding of these fusion proteins to JNK1 wassimilar to the level of binding to JNK2. The binding of JNK to JIP-1 wassignificantly greater than the binding of JNK to ATF2 or c-Jun. Controlexperiments demonstrated that JIP-1 did not bind to p38 MAP kinase.These data establish that JIP-1 binding to JNK1 is quantitativelysimilar to JIP-1 binding to JNK2, and that JNK binding to JIP-1 issignificantly greater than JNK binding to the transcription factors ATF2and c-Jun.

[0162] The GST-JIP-1 fusion proteins containing various portions of theJIP-1 gene were used to define regions of JIP-1 that are required forJNK interaction. A GST-JIP-1 fusion protein containing residues 127-281of JIP-1 bound both JNK1 and JNK2. No JNK binding was detected inexperiments using the central region (residues 164-240) or the carboxyterminal region (residues 203-281) of JIP-1. However, JNK bindingactivity was observed in experiments using the amino terminal region(residues 127-202 of JIP-1). These data indicate that residues 127-164of JIP-1 are required for JNK binding activity.

[0163] To more precisely define the JIP-1 sequence required for JNKbinding activity, additional GST-JNK fusion proteins; were constructed.These fusion proteins contained residues 135-202 (SEQ ID NO:18), 144-202(SEQ ID NO:19), 154-202 (SEQ ID NO:20), 164-202 (SEQ ID NO:21), 127-143(SEQ ID NO:22), 127-153 (SEQ ID NO:23), or 127-163 (SEQ ID NO:24, ofJIP-1. Proteins containing residues 127-202, 135-202, 144-202, 154-202,and 127-163 all bound both JNK1 and JNK2. Thus, JIP-1 residues 144-163are important for the interaction of JIP-1 with JNK. As shown in FIG. 2,this region of JIP-1 shares sequence similarity with the JNK bindingdomains of ATF2 and c-Jun.

[0164] This assay can also be used to analyze a given polypeptide todetermine whether it is, or is not, a JIP-1 polypeptide.

Example 4 A Small NH₂-terminal Region of JIP-1 is Sufficient forInteraction with JNK

[0165] The effect of increasing concentrations (0, 4, 8, 16, 32, and 64μg/ml) of a synthetic peptide corresponding to JIP-1 residues 148-174(SEQ ID NO:3) or a control peptide having a scrambled sequence (SEQ IDNO:12) on JIP-1-JNK interaction was examined. Peptides were synthesizedusing an Applied Biosystems machine. Cell lysates containing Flagepitope-tagged JNK were incubated with GST-JIP-1 (residues 127-281)prebound to glutathione-Sepharose. The beads were washed, and boundproteins were detected by protein immunoblot analysis with aFlag-specific antibody. While the control peptide had no effect,incubation with the synthetic peptide corresponding to residues 148-174of JIP-1 resulted in a dose-dependent decrease in JIP-1 binding to JNK1,indicating that this peptide competes with JIP-1 for binding to JNK.

[0166] The JNK binding domain of JIP-1 contains three amino eacids,Lys-155, Thr-159, and Leu-160, that are conserved in the JNK bindingdomains of ATF2 and c-Jun (FIG. 2). A hydrophobic amino acid, Leu, isfound at residue 162 of JIP-1. ATF2 and c-Jun also contain hydrophobicamino acids (ATF2, Phe; c-Jun, Leu) in the position corresponding toresidue 162 of JIP-1. To test if these conserved residues are involvedin JNK binding, the wild type JIP-1 JBD (residues 148-174) wassubstituted with glycines in these positions to produce the peptidesshown in FIG. 3 (SEQ ID NOs:7-11). The mutant peptides are identical tothe wild type peptide except for the indicated glycine substitutions.The binding of JNK1 to GST, as well as a GST fusion protein containingJIP-1 residues 127-281, was examined in the absence and presence of thesynthetic peptides (64 μg/ml). A peptide with a scrambled sequence (SEQID NO:12) was used as a control. The peptide representing the wild-typeJIP-1 sequence caused a dose-dependent inhibition of JIP-1 binding toJNK. In contrast, the control peptide caused no change in JNK binding.When any of the glycine-substituted peptides was used in the assay, theinhibition of JNK binding was greatly reduced compared to that observedwith the wild type peptide. These data indicate that Lys-155, Thr-159,Leu-160, and Leu-162 are involved in JIP-1 binding to JNK.

Example 5 JIP-1 is a Selective Inhibitor of JNK Activity

[0167] To test whether JIP-1 competes with Jun and ATF2 for interactionwith JNK, the effect of JIP-1 on transcription factor-mediatedphosphorylation of exogenous substrates was analyzed using an in vitroprotein kinase assay. In these experiments, Chinese hamster ovary (CHO)cells were serum-starved for one hour. In some experiments, the cellswere treated with 10 ng/ml mouse interleukin 1 or 100 nM phorbolmyristic acetate. JNK, p38, and ERK protein kinase activity was measuredin an immune complex kinase assay using 3 μg of the substrates GST-Jun,GST-ATF2, and myelin basic protein (MBP), respectively. Protein kinaseassays were performed using 40 μl 20 mM Hepes (pH 7.4), 20 mM MgCl_(2,)20 mM β-glycerophosphate, 2 mM dithiothreitol, 0.1 mM sodiumorthovanadate, 50 μM [γ-³²P]ATP (10 Ci/mmol). After incubation for 30minutes at 30° C., phosphorylation of substrates was analyzed bypolyacrylamide gel electrophoresis and autoradiography.

[0168] The results show that JIP-1 markedly inhibited thephosphorylation of c-Jun by JNK. However, JIP-1 caused no significantchange in the phosphorylation of substrates by the related MAP kinasesp38 and ERK2. JIP-1 is thus a selective inhibitor of JNK.

[0169] This same assay can be used to determine whether a specificpolypeptide has the same phosphorylation function of wildtype JIP-1.

Example 6 Expression of JIP-1 Inhibits Targets of the JNK-regulatedSignal Transduction Pathway

[0170] The effect of JIP-1 on targets of the JNK signal ransductionpathway, including the transcription factors c-Jun, ATF2 and Elk-1, wasexamined to determine whether JIP-1 inhibits signal transduction by JNK.

[0171] In these experiments, CHO cells were cotransfected withconstructs encoding the transcription factors and JIP-1 using previouslydescribed methods. Dérijard et al., supra; Sluss et al., supra; Gupta etal., supra. A luciferase reporter plasmid was used to monitor theexpression of the transcription factors. Transfection efficiency wasdetermined using a β-galactosidase expression vector. Constructs used inthese experiments included GAL4 fusions with the c-myc, Sp1 and VP16activation domains (described in Whitmarsh et al., Science, 269:403(1995); Davis, Science, 269:403 (1995); Gille et al., Curr. Biol.,5:1191 (1995)). Other constructs used were GAL4-c-Jun, in which GAL4 isfused to c-Jun; GAL4-c-Jun (S63A/S73A), in which GAL4 is fused to amutant c-Jun in which the serines at positions 63 and 73 have beenchanged to alanines; GAL4-ATF2, in which GAL4 is fused to wild type ATF2(ATF2(Thr-69, 71)); GAL4-ATF2 (T69A/T71A) (ATF2(Ala-69/71)), in whichGAL4 has been fused to a mutant ATF2 in which the tyrosines at positions69 and 71 have been changed to alanines; GAL4-Elk-1, in which GAL4 hasbeen fused to Elk-1; and GAL4-Elk-1 (S383A), in which GAL4 has beenfused to a mutant Elk-1 in which the serine at position 383 has beenchanged to an alanine. Gupta et al., EMBO J, 15:2760-2770 (1996);Raingeaud et al., Mol. Cell. Biol., 16:1247-1255 (1996); Whitmarsh etal., Science, 269:403-407 (1995).

[0172] Insertional overlapping PCR, described in detail in Ho, supra,was used to construct expression vectors for the JNK binding domain(JBD; amino acid residues 127-281) of JIP-1 and for a mutant JIP-1lacking the SH3 (amino acid residues 491-540) domain.

[0173] Transfected cells were activated by treatment with 10% (v/v)fetal calf serum, and luciferase and β-galactosidase activities weremeasured in cell lysates at 48 hours post-transfection. The results areshown in FIGS. 4A-4C. The data are presented as the ratio of luciferaseactivity (light units) to β-galactosidase activity (OD units) measuredin the cell extracts (mean±SEM (n=3)), and are normalized to theluciferase activity detected in the absence of JBD (FIGS. 4A and 4B) orATF2 (FIG. 4C).

[0174] Control experiments demonstrated that the JNK binding domain(JBD; residues 127-281 of JIP-1) did not inhibit reporter geneexpression mediated by the activation domains of c-Myc, E1a, Sp1, andVP16 (FIG. 4A). Significant inhibition of c-Jun and ATF2 transcriptionalactivity by JIP-1 was observed, however (FIG. 4B). The partialinhibition of Elk-1 transcriptional activity shown in FIG. 4B mayreflect an association of both ERK and JNK MAP kinases with Elk-1regulation. Mutation of the JNK phosphorylation sites in ATF2, c-Jun,and Elk-1 caused lower basal transcriptional activity that was notmarkedly inhibited by JIP-1.

[0175] As shown in FIG. 4C, inhibition of JNK-regulated gene expressionwas observed in experiments using wild-type JIP-1, the JNK bindingdomain (JBP), and ΔSH3, a JIP-1 deletion mutant lacking the SH3 domain.Together, these data indicate that JIP-1 suppresses JNK-regulated geneexpression, and that the JNK binding domain is sufficient for thisactivity.

[0176] This assay can be used to determine whether specific polypeptideshave the same effect on signal transduction as full length, wildtypeJIP-1.

Example 7 Subcellular Distribution of JIP-1

[0177] The subcellular distribution of JIP-1 was analyzed by performingindirect immunofluorescence on JIP-1-expressing cells. In theseexperiments, the cells were grown on coverslips, fixed with 4%paraformaldehyde, permeabilized with 0.25% Triton X-100, and processedfor immunofluorescence microscopy. JIP-1 was detected in the cytoplasm,but not the nucleus, of control and UV-irradiated cells. In contrast,JNK is detected in both the cytoplasmic and nuclear compartments.

[0178] It is likely that it is JNK in the nucleus which is involved inthe regulation of gene expression. Since JIP-1 is cytoplasmic, it wasunclear how it could inhibit the nuclear function of JNK. To investigatethe mechanism of JIP-1 action, the distribution of JNK was examined incells that had been transfected with either hemagglutinin (HA)-taggedJNK1 (HA-JNK1) alone, or HA-JNK1 and Flag-tagged JIP-1 (Flag-JIP-1). Inthese experiments, the cells were exposed to a potent JNK activator (40J/m² UV-C) for one hour prior to processing for immunofluorescence. Theprimary antibodies used were rabbit anti-HA (12CA5; BoehringerMannheim), which recognizes HA-tagged JNK1; and mouse monoclonalanti-Flag (M2; IBI-Kodak), which recognizes Flag-tagged JIP-1. Thesecondary antibodies were Texas Red-goat anti-mouse Ig and fluorosceinisothiocyanate-conjugated donkey anti-rabbit Ig (JacksonImmunoresearch). Procedures for digital imaging microscopy and imagerestoration using the exhaustive photon reassignment algorithm aredescribed in Carrington et al., Science, 268:1483 (1995). Individualoptical sections were inspected using computer graphics software on aSilicon Graphics workstation.

[0179] The results demonstrate that expression of JIP-1 reduces theamount of nuclear JNK detected in control and UV-irradiated cells. Incontrast, JIP-1 expression has no significant effect on the subcellulardistribution of p38 MAP kinase, which is also located in both nuclearand cytoplasmic compartments of cultured cells. These data indicate thatJIP-1 expression results in selective cytoplasmic retention of JNK. Itis likely that this cytoplasmic retention contributes to the ability ofJIP-1 to inhibit JNK-mediated signal transduction.

Example 8 JIP-1 Inhibits Nerve Growth Factor Withdrawal-InducedApoptosis

[0180] The effect of JIP-1 on this biological response was investigatedto determine whether JIP-1 inhibits the biological effects of the JNKsignaling pathway. NGF withdrawal-induced apoptosis of differentiatedPC12 cells was examined following transfection with the expressionvectors pcDNA3 (control) and pcDNA3-Flag-JIP-1 (containing residues127-281 of JIP-1) using methods described previously. Xia et al.,Science, 270:1326 (1995). The percentage of apoptotic cells in the totaltransfected cell population was blind-scored and quantitated. This assayscores adherent cells with an apoptotic morphology at 17 hours followingNGF withdrawal. Cells that have completed apoptosis are nonadherent andare not scored. Thus, while the cumulative extent of apoptosis is large(100%), lower numbers of apoptotic cells are detected at a single timepoint.

[0181] The results are shown in FIG. 5. The data shown arerepresentative of three independent experiments. The error bars in thegraph indicate the SEM and the numbers within each bar are the totalnumber of transfected cells counted. Expression of the JNK bindingdomain of JIP-1 (JBD; residues 127-281) in differentiated PC12 cellsmarkedly reduced apoptosis following NGF-withdrawal. These datademonstrate that JIP-1 suppresses JNK-mediated signal transduction. Thisassay can be used to test new JIP-1 polypeptides.

Example 9 JIP-1 Inhibits Pre-B Cell Transformation by BCR-ABL

[0182] The BCR-ABL oncogene, which is associated with chronicmyelogenous leukemia (CML), causes JNK activation in the absence ofincreased ERK activity. Raitano et al., Proc. Natl. Acad. Sci. USA,92:11746 (1995). Oncogenic transformation by BCR-ABL may be mediated inpart by the JNK signaling pathway. Since JIP-1 can inhibit JNK activity,the effect of JIP-1 expression on oncogenic transformation was examined.Plasmid vectors expressing v-ABL or BCR-ABL and Flag-tagged JBD(residues 127-281) of JIP-1 were used to transfect 293 cells. JNKactivity was then measured in an immune complex kinase assay of lysatesof the transfected 293 cells, using a polyclonal JNK antibody andGST-Jun as the substrate. Control experiments demonstrated that v-ABLand BCR-ABL caused constitutive JNK activation (approximately 5-fold),which was blocked by co-expression of the JBD of JIP-1. JIP-1 cantherefore inhibit BCR-ABL-mediated activation of JNK.

[0183] To examine the effect of JIP-1 on BCR-ABL-mediated cellulartransformation, bone marrow transformation assays were performed usingrecombinant retroviruses packaged with 293T cells as described inSawyers et al., J. Exp. Med., 181:307 (1995). Bicistronic retrovirusesexpressing BCR-ABL, alone, or in combination with the Flag-tagged JBD(residues 127-281) of JIP-1, were prepared by subcloning p185BCR-ABLinto the ClaI site of pSRαTK, downstream of the internal TK promoter, tocreate pSRαMSVTKp185. The JBD was subcloned into the upstream EcoRI sitein the sense and antisense orientations. The structures of theretroviral constructs are shown in FIG. 6.

[0184] Transfection of 293 cells with these retroviral constructsresulted in expression of the appropriate proteins, as demonstrated byimmunoblot analysis of whole cell lysates using antibodies specific forABL to detect BCR-ABL, and antibodies to Flag to detect the JBD ofJIP-1. The recombinant retroviruses were then used to infect primarymouse marrow cells, and the transformation of pre-B cells was monitoredin culture. FIG. 6 shows the mean density (±SE) of non-adherent pre-Bcells on day 10 post infection. The data shown are derived from threeindependent experiments plated in triplicate. As expected, BCR-ABLcaused pre-B cell transformation. The JBD of JIP-1 caused markedinhibition of transformation when present in the sense, but not theanti-sense, orientation. In some cultures infected with BCR-ABL and JBD,pre-B cell outgrowths were detected after 3-4 weeks, but these clonesdemonstrated no expression of JBD by protein immunoblot analysis. SinceJIP-1 inhibits JNK signaling, these results indicate that the JNKpathway is required for pre-B cell transformation by BCR-ABL.

[0185] The demonstration that JNK is involved in both apoptosis andoncogenic transformation indicates that the biological actions of theJNK signal transduction pathway depend on the specific cellular context.The integration of JNK with other signal transduction pathways may be animportant determinant of the functional consequences of JNK activation.The ability of JIP-1, an inhibitor of the JNK signal transductionpathway, to block both transformation and apoptosis is consistent withthis hypothesis.

[0186] The physiological function of JIP-1 may be to suppress signaltransduction by the JNK pathway. For example, JIP-1 may compete withsubstrates that bind JNK. Alternatively, JIP-1 may have a more directrole in targeting JNK to specific regions of the cell or to specificsubstrates. Since JIP-1 causes redistribution of JNK within the cell,JIP-1 may function as a cytoplasmic anchor for JNK. The tethering of JNKin the cytoplasm by interactions with JIP-1 provides a mechanism forcontrolling signal transduction by the JNK pathway, and the relatedphenomena of apoptosis and transformation.

[0187] This assay can be used to determine if specific polypeptides havethe same effect on cellular transformation as full length, wildtypeJIP-1.

Example 10 Screening for Peptides with JIP-1 Activity

[0188] Peptides suspected of having JIP-1 activity can be tested in theJNK binding assay described supra. Peptides are synthesized by methodsthat are well known to those skilled in the art; for example, using anApplied Biosystems synthesizer. Cell lysates containing Flagepitope-tagged JNK are incubated with GST-JIP-1 (residues 127-281 boundto glutathione-Sepharose, either with or without synthetic peptides (0,4, 8, 16, 32 or 64 μg/ml), for one hour at 4° C. The beads are washed inTLB, and the bound proteins are detected by protein immunoblot analysiswith a Flag-specific antibody, e.g., M2. Synthetic peptides with JIP-1activity are those which inhibit the interaction of JIP-1 with JNK, asdetected in this assay. These synthetic peptides should possess at least60% of the binding activity of JIP-1.

Example 11 Therapeutic Applications

[0189] JIP-1 is shown herein to be capable of inhibiting apoptosis andtransformation. Compositions containing JIP-1 polypeptides or nucleicacids can be administered to treat conditions characterized by thesephenomena. Nucleic acids can be administered by, methods described in,e.g., Ausubel, et al., supra. A standard dosage would be from 1 to 1000μg/kg of body weight. Polypeptides, peptides and peptide mimetics of theinvention can be formulated according to procedures which are well knownto those skilled in the art. A standard dosage of polypeptide also wouldbe from 1 to 1000, e.g., 10 to 500, or 20 to 200 μg/kg of body weight.

[0190] Animal models for testing the effect of JIP-1 therapeuticcompositions include the bcr-abl leukemia model. Daley et al., Science,247:824-830 (1990). Other animal models that can be used include themyc/ras transformation model. Sinn et al., Cell, 49:465-475 (1987).Animal models for testing the effect of JIP-1 therapeutic compositionson conditions associated with apoptosis include a model of excitotoxicstress in the hippocampus, and a model of E2F-1 induced apoptosis.Ben-Ari et al., Neuroscience, 14:375-403 (1985); Field et al., Cell,85:549-561 (1996). Other conditions that can be treated with JIP-1compositions include liver damage (Mendelson et al., Proc. Natl. Acad.Sci. USA, 93:12908-12913 (1996)); kidney disease and organtransplantation (DeMari et al., Am. J. Physiol., 272:F292-F298 (1997));and heart disease (Pombo et al., J. Biol. Chem., 269:26546-26551 (1994);Force et al., Circ. Res., 78:947-953 (1996).

Example 12 Diagnostic Applications

[0191] The polypeptides and antibodies of the invention can be used todetect or monitor JIP-1 expression. Levels of JIP-1 protein in a samplecan be assayed by any standard technique. For example, JIP-1 proteinexpression can be monitored by standard immunological orimmunohistochemical procedures using the antibodies described herein.See, e.g., Ausubel et al., supra; Bancroft et al., Theory and Practiceof Histological Techniques, Churchill Livingstone (1982). Alternatively,JIP-1 expression can be assayed by standard Northern blot analysis, orcan be aided by PCR. See Ausubel, supra; Ehrlich, ed., PCR Technology:Principles and Applications for DNA Amplification, Stockton Press, NewYork. Point mutations in the JIP-1 sequence can be detected using wellknown nucleic acid mismatch detection techniques. Lower than normallevels of JIP-1 would have the effect of altering apoptosis, whilehigher than normal levels would cause immune suppression, alteredinflammatory responses, and alterations in tumor growth.

Other Embodiments

[0192] The invention also includes naturally occurring andnon-naturally-occurring allelic variants of JIP-1. Compared to the mostcommon naturally occurring nucleotide sequence encoding JIP-1, thenucleic acid sequence encoding allelic variants may have a substitution,deletion, or addiction of one or more nucleotides. The preferred allelicvariants are functionally equivalent to naturally occurring JIP-1.

[0193] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, that theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages and modifications are within the scope of thefollowing claims.

1 30 660 amino acids amino acid linear protein internal JIP-1 protein 1Met Ala Glu Arg Glu Ser Gly Leu Gly Gly Gly Ala Ala Ser Pro Pro 1 5 1015 Ala Ala Ser Pro Phe Leu Gly Leu His Ile Ala Ser Pro Pro Asn Phe 20 2530 Arg Leu Thr His Asp Ile Ser Leu Glu Glu Phe Glu Asp Glu Asp Leu 35 4045 Ser Glu Ile Thr Asp Glu Cys Gly Ile Ser Leu Gln Cys Lys Asp Thr 50 5560 Leu Ser Leu Arg Pro Pro Arg Ala Gly Leu Leu Ser Ala Gly Ser Ser 65 7075 80 Gly Ser Ala Gly Ser Arg Leu Gln Ala Glu Met Leu Gln Met Asp Leu 8590 95 Ile Asp Ala Ala Gly Asp Thr Pro Gly Ala Glu Asp Asp Glu Glu Glu100 105 110 Glu Asp Asp Glu Leu Ala Ala Gln Arg Pro Gly Val Gly Pro ProLys 115 120 125 Ala Glu Ser Asn Gln Asp Pro Ala Pro Arg Ser Gln Gly GlnGly Pro 130 135 140 Gly Thr Gly Ser Gly Asp Thr Tyr Arg Pro Lys Arg ProThr Thr Leu 145 150 155 160 Asn Leu Phe Pro Gln Val Pro Arg Ser Gln AspThr Leu Asn Asn Asn 165 170 175 Ser Leu Gly Lys Lys His Ser Trp Gln AspArg Val Ser Arg Ser Ser 180 185 190 Ser Pro Leu Lys Thr Gly Glu Gln ThrPro Pro His Glu His Ile Cys 195 200 205 Leu Ser Asp Glu Leu Pro Pro GlnGly Ser Pro Val Pro Thr Gln Asp 210 215 220 Arg Gly Thr Ser Thr Asp SerPro Cys Arg Arg Ser Ala Ala Thr Gln 225 230 235 240 Met Ala Pro Pro SerGly Pro Pro Ala Thr Ala Pro Gly Gly Arg Gly 245 250 255 His Ser His ArgAsp Arg Ile His Tyr Gln Ala Asp Val Arg Leu Glu 260 265 270 Ala Thr GluGlu Ile Tyr Leu Thr Pro Val Gln Arg Pro Pro Asp Pro 275 280 285 Ala GluPro Thr Ser Thr Phe Met Pro Pro Thr Glu Ser Arg Met Ser 290 295 300 ValSer Ser Asp Pro Asp Pro Ala Ala Tyr Ser Val Thr Ala Gly Arg 305 310 315320 Pro His Pro Ser Ile Ser Glu Glu Asp Glu Gly Phe Asp Cys Leu Ser 325330 335 Ser Pro Glu Arg Ala Glu Pro Pro Gly Gly Gly Trp Arg Gly Ser Leu340 345 350 Gly Glu Pro Pro Pro Pro Pro Arg Ala Ser Leu Ser Ser Asp ThrSer 355 360 365 Ala Leu Ser Tyr Asp Ser Val Lys Tyr Thr Leu Val Val AspGlu His 370 375 380 Ala Gln Leu Glu Leu Val Ser Leu Arg Pro Cys Phe GlyAsp Tyr Ser 385 390 395 400 Asp Glu Ser Asp Ser Ala Thr Val Tyr Asp AsnCys Ala Ser Ala Ser 405 410 415 Ser Pro Tyr Glu Ser Ala Ile Gly Glu GluTyr Glu Glu Ala Pro Gln 420 425 430 Pro Arg Pro Pro Thr Cys Leu Ser GluAsp Ser Thr Pro Asp Glu Pro 435 440 445 Asp Val His Phe Ser Lys Lys PheLeu Asn Val Phe Met Ser Gly Arg 450 455 460 Ser Arg Ser Ser Ser Ala GluSer Phe Gly Leu Phe Ser Cys Val Ile 465 470 475 480 Asn Gly Glu Glu HisGlu Gln Thr His Arg Ala Ile Phe Arg Phe Val 485 490 495 Pro Arg His GluAsp Glu Leu Glu Leu Glu Val Asp Asp Pro Leu Leu 500 505 510 Val Glu LeuGln Ala Glu Asp Tyr Trp Tyr Glu Ala Tyr Asn Met Arg 515 520 525 Thr GlyAla Arg Gly Val Phe Pro Ala Tyr Tyr Ala Ile Glu Val Thr 530 535 540 LysGlu Pro Glu His Met Ala Ala Leu Ala Lys Asn Ser Cys Val Leu 545 550 555560 Glu Ile Ser Val Arg Gly Val Lys Ile Gly Val Lys Ala Asp Asp Ala 565570 575 Leu Glu Ala Lys Gly Asn Lys Cys Ser His Phe Phe Gln Leu Lys Asn580 585 590 Ile Ser Phe Cys Gly Tyr His Pro Lys Asn Asn Lys Tyr Phe GlyPhe 595 600 605 Ile Thr Lys His Pro Ala Asp His Arg Phe Ala Cys His ValPhe Val 610 615 620 Ser Glu Asp Ser Thr Lys Ala Leu Ala Glu Ser Val GlyArg Ala Phe 625 630 635 640 Gln Gln Phe Tyr Lys Gln Phe Val Glu Tyr ThrCys Pro Thr Glu Asp 645 650 655 Ile Tyr Leu Glu 660 2832 base pairsnucleic acid double linear cDNA Coding Sequence 180...2159 JIP-1 cDNA 2CTCGAGGTCG ACGGTATCGA TAAGCTTGAT ATCGCTGTCC GGAGCCGCGC CCGCCCCAGC 60TCAGTCCGAA CCCCGCGGCG GCGGCGGCCT CCTCCACGCG CCTCCGCTGC TGCCGCCGCC 120GCCGCCGCCG CCGCCTCCCG CGCCGCTCTC CGCCCGGATG GCCAGGGCTG CACCCCGGA 179 ATGGCG GAG CGA GAG AGC GGC CTG GGC GGG GGC GCC GCG TCC CCA CCG 227 Met AlaGlu Arg Glu Ser Gly Leu Gly Gly Gly Ala Ala Ser Pro Pro 1 5 10 15 GCCGCT TCC CCA TTC CTG GGA CTG CAC ATC GCG TCG CCT CCC AAT TTC 275 Ala AlaSer Pro Phe Leu Gly Leu His Ile Ala Ser Pro Pro Asn Phe 20 25 30 AGG CTCACC CAT GAC ATC AGC CTG GAG GAG TTT GAG GAT GAA GAC CTT 323 Arg Leu ThrHis Asp Ile Ser Leu Glu Glu Phe Glu Asp Glu Asp Leu 35 40 45 TCG GAG ATCACT GAC GAG TGT GGC ATC AGC CTG CAG TGC AAA GAC ACC 371 Ser Glu Ile ThrAsp Glu Cys Gly Ile Ser Leu Gln Cys Lys Asp Thr 50 55 60 CTG TCT CTC CGGCCC CCG CGC GCC GGG CTG CTG TCT GCG GGT AGC AGC 419 Leu Ser Leu Arg ProPro Arg Ala Gly Leu Leu Ser Ala Gly Ser Ser 65 70 75 80 GGC AGC GCG GGGAGC CGG CTG CAG GCG GAG ATG CTG CAG ATG GAC CTG 467 Gly Ser Ala Gly SerArg Leu Gln Ala Glu Met Leu Gln Met Asp Leu 85 90 95 GTC GAC GCG GCA GGTGAC ACT CCG GGC GCC GAG GAC GAC GAG GAG GAG 515 Ile Asp Ala Ala Gly AspThr Pro Gly Ala Glu Asp Asp Glu Glu Glu 100 105 110 GAG GAC GAC GAG CTCGCT GCC CAA CGA CCA GGA GTG GGG CCT CCC AAA 563 Glu Asp Asp Glu Leu AlaAla Gln Arg Pro Gly Val Gly Pro Pro Lys 115 120 125 GCG GAG TCC AAC CAGGAT CCG GCG CCT CGC AGC CAG GGC CAG GGC CCG 611 Ala Glu Ser Asn Gln AspPro Ala Pro Arg Ser Gln Gly Gln Gly Pro 130 135 140 GGC ACA GGC AGC GGAGAC ACC TAC CGA CCC AAG AGG CCT ACC ACG CTC 659 Gly Thr Gly Ser Gly AspThr Tyr Arg Pro Lys Arg Pro Thr Thr Leu 145 150 155 160 AAC CTT TTC CCGCAG GTG CCG CGG TCT CAG GAC ACG CTG AAT AAT AAC 707 Asn Leu Phe Pro GlnVal Pro Arg Ser Gln Asp Thr Leu Asn Asn Asn 165 170 175 TCT TTA GGC AAAAAG CAC AGT TGG CAG GAC CGT GTG TCT CGA TCA TCC 755 Ser Leu Gly Lys LysHis Ser Trp Gln Asp Arg Val Ser Arg Ser Ser 180 185 190 TCC CCT CTG AAGACA GGA GAA CAG ACG CCT CCA CAT GAA CAC ATC TGC 803 Ser Pro Leu Lys ThrGly Glu Gln Thr Pro Pro His Glu His Ile Cys 195 200 205 CTG AGT GAT GAGCTG CCA CCC CAG GGC AGT CCT GTT CCC ACC CAG GAC 851 Leu Ser Asp Glu LeuPro Pro Gln Gly Ser Pro Val Pro Thr Gln Asp 210 215 220 CGC GGC ACT TCCACC GAC AGC CCT TGT CGC CGA AGT GCA GCC ACC CAG 899 Arg Gly Thr Ser ThrAsp Ser Pro Cys Arg Arg Ser Ala Ala Thr Gln 225 230 235 240 ATG GCA CCTCCA AGC GGT CCC CCT GCC ACT GCT CCT GGT GGC CGG GGC 947 Met Ala Pro ProSer Gly Pro Pro Ala Thr Ala Pro Gly Gly Arg Gly 245 250 255 CAC TCC CATCGA GAC CGA ATC CAC TAC CAG GCA GAT GTG CGG CTC GAG 995 His Ser His ArgAsp Arg Ile His Tyr Gln Ala Asp Val Arg Leu Glu 260 265 270 GCG ACT GAGGAG ATC TAC CTG ACC CCA GTG CAG AGG CCC CCA GAC CCT 1043 Ala Thr Glu GluIle Tyr Leu Thr Pro Val Gln Arg Pro Pro Asp Pro 275 280 285 GCA GAA CCCACC TCC ACC TTC ATG CCA CCC ACG GAG AGC CGG ATG TCA 1091 Ala Glu Pro ThrSer Thr Phe Met Pro Pro Thr Glu Ser Arg Met Ser 290 295 300 GTT AGC TCGGAT CCA GAC CCT GCC GCT TAC TCT GTA ACT GCG GGG CGG 1139 Val Ser Ser AspPro Asp Pro Ala Ala Tyr Ser Val Thr Ala Gly Arg 305 310 315 320 CCA CACCCC TCC ATC AGT GAA GAG GAT GAG GGC TTC GAC TGC CTG TCA 1187 Pro His ProSer Ile Ser Glu Glu Asp Glu Gly Phe Asp Cys Leu Ser 325 330 335 TCC CCAGAG CGA GCT GAG CCA CCA GGT GGA GGG TGG CGG GGA AGC CTC 1235 Ser Pro GluArg Ala Glu Pro Pro Gly Gly Gly Trp Arg Gly Ser Leu 340 345 350 GGG GAGCCA CCA CCG CCT CCA CGG GCC TCA CTG AGC TCG GAC ACC AGC 1283 Gly Glu ProPro Pro Pro Pro Arg Ala Ser Leu Ser Ser Asp Thr Ser 355 360 365 GCA CTGTCC TAC GAC TCG GTC AAG TAC ACA CTG GTG GTG GAT GAA CAT 1331 Ala Leu SerTyr Asp Ser Val Lys Tyr Thr Leu Val Val Asp Glu His 370 375 380 GCC CAGCTT GAG TTG GTG AGC CTG CGG CCG TGC TTT GGA GAT TAC AGT 1379 Ala Gln LeuGlu Leu Val Ser Leu Arg Pro Cys Phe Gly Asp Tyr Ser 385 390 395 400 GACGAA AGC GAC TCT GCC ACT GTC TAT GAC AAC TGT GCC TCT GCC TCC 1427 Asp GluSer Asp Ser Ala Thr Val Tyr Asp Asn Cys Ala Ser Ala Ser 405 410 415 TCGCCC TAC GAG TCA GCC ATT GGT GAG GAG TAT GAG GAG GCC CCT CAG 1475 Ser ProTyr Glu Ser Ala Ile Gly Glu Glu Tyr Glu Glu Ala Pro Gln 420 425 430 CCCCGG CCT CCC ACC TGC CTC TCA GAG GAC TCC ACC CCG GAT GAG CCT 1523 Pro ArgPro Pro Thr Cys Leu Ser Glu Asp Ser Thr Pro Asp Glu Pro 435 440 445 GATGTC CAC TTC TCT AAG AAG TTT CTG AAT GTC TTC ATG AGT GGC CGC 1571 Asp ValHis Phe Ser Lys Lys Phe Leu Asn Val Phe Met Ser Gly Arg 450 455 460 TCTCGT TCC TCC AGT GCT GAG TCC TTT GGG CTG TTC TCC TGC GTC ATC 1619 Ser ArgSer Ser Ser Ala Glu Ser Phe Gly Leu Phe Ser Cys Val Ile 465 470 475 480AAT GGG GAG GAG CAT GAG CAA ACC CAT CGG GCT ATA TTC AGG TTT GTG 1667 AsnGly Glu Glu His Glu Gln Thr His Arg Ala Ile Phe Arg Phe Val 485 490 495CCT CGG CAT GAA GAT GAA CTT GAG CTG GAA GTG GAT GAC CCC CTG CTG 1715 ProArg His Glu Asp Glu Leu Glu Leu Glu Val Asp Asp Pro Leu Leu 500 505 510GTG GAG CTG CAG GCA GAA GAC TAT TGG TAT GAG GCC TAT AAC ATG CGC 1763 ValGlu Leu Gln Ala Glu Asp Tyr Trp Tyr Glu Ala Tyr Asn Met Arg 515 520 525ACC GGA GCC CGC GGG GTC TTC CCT GCC TAC TAT GCC ATT GAG GTC ACC 1811 ThrGly Ala Arg Gly Val Phe Pro Ala Tyr Tyr Ala Ile Glu Val Thr 530 535 540AAG GAG CCT GAG CAC ATG GCA GCC CTT GCC AAA AAC AGC TGT GTC CTT 1859 LysGlu Pro Glu His Met Ala Ala Leu Ala Lys Asn Ser Cys Val Leu 545 550 555560 GAG ATC AGT GTC AGG GGT GTC AAG ATA GGC GTC AAA GCT GAT GAT GCT 1907Glu Ile Ser Val Arg Gly Val Lys Ile Gly Val Lys Ala Asp Asp Ala 565 570575 CTG GAG GCC AAG GGA AAT AAA TGT AGC CAC TTC TTC CAG CTA AAG AAC 1955Leu Glu Ala Lys Gly Asn Lys Cys Ser His Phe Phe Gln Leu Lys Asn 580 585590 ATC TCT TTC TGT GGA TAC CAT CCA AAG AAT AAC AAG TAC TTT GGG TTT 2003Ile Ser Phe Cys Gly Tyr His Pro Lys Asn Asn Lys Tyr Phe Gly Phe 595 600605 ATC ACT AAG CAC CCT GCT GAC CAC CGG TTT GCC TGC CAT GTC TTT GTG 2051Ile Thr Lys His Pro Ala Asp His Arg Phe Ala Cys His Val Phe Val 610 615620 TCT GAA GAT TCC ACC AAA GCC CTG GCG GAG TCT GTG GGG CGT GCA TTT 2099Ser Glu Asp Ser Thr Lys Ala Leu Ala Glu Ser Val Gly Arg Ala Phe 625 630635 640 CAG CAG TTC TAC AAG CAG TTT GTG GAG TAT ACC TGT CCT ACA GAA GAT2147 Gln Gln Phe Tyr Lys Gln Phe Val Glu Tyr Thr Cys Pro Thr Glu Asp 645650 655 ATC TAC TTG GAG TAGCAGCACC CCCACTCTCT GCAGCCCCTC AGCCCCAAGC 2199Ile Tyr Leu Glu 660 CAGTGCAAGG ACAGCTGGCT GCTGACAGGA TGTGGTACTGCCACAAAAGA ATGGGGGAAT 2259 GAGGGCTGTT GGGTCGGGGG GGCCGGGGTT TGGGGAGAGGCAGATGCAGT TTATTGTAAT 2319 ATATGGGGTT AGATTAATCT ATGGAGGACA GTACAGGCTCTCTGGGGACT GGGGAAGGGT 2379 GGGGCTGGGG GGTGGGGGGT CAGGCCCCTG GCCACAGAGGGACTCCCTAG GAACAGAGGC 2439 ACTGTCCCAT CCTGGGCCTG TTTCATGCTA GGGGCCCTGGCTTTCTGGCT CTTGGCTCCT 2499 GCCTTGACAA AGCCCATGCC ACCTGGAAGT GTCCAGCTTCCCTTGTCCCC ACCTTGACCG 2559 GAGCCCTGAG CTCAGGCTGA GCCCACGCAC CTCCAAAGGACTTTCCAGTA AGGAAATGGC 2619 AACGTGTGAC CGTGGAGACC CTGTTCTCAT CTGTGGGGCCTCTGGGCAGC TCCAACCTCC 2679 AGCCTGGCTA GCACACAGGT CCTCGCAAGG TTGTGTGTGCAAGGAGAGGG CCACAGTAAG 2739 CCCCATCTGC CAGGAAAAGG AGGCCTCTTA GCTGGCCCCAGCCAGCCGGT CACTGTCTTG 2799 TCACCTGGCT ACTATTAAAG TGCCATCTCG TGC 2832 27amino acids amino acid linear peptide JIP-1 protein, amino acids 148-1743 Ser Gly Asp Thr Tyr Arg Pro Lys Arg Pro Thr Thr Leu Asn Leu Phe 1 5 1015 Pro Gln Val Pro Arg Ser Gln Asp Thr Leu Asn 20 25 155 amino acidsamino acid linear protein JIP-1 protein, amino acids 127-281 4 Pro LysAla Glu Ser Asn Gln Asp Pro Ala Pro Arg Ser Gln Gly Gln 1 5 10 15 GlyPro Gly Thr Gly Ser Gly Asp Thr Tyr Arg Pro Lys Arg Pro Thr 20 25 30 ThrLeu Asn Leu Phe Pro Gln Val Pro Arg Ser Gln Asp Thr Leu Asn 35 40 45 AsnAsn Ser Leu Gly Lys Lys His Ser Trp Gln Asp Arg Val Ser Arg 50 55 60 SerSer Ser Pro Leu Lys Thr Gly Glu Gln Thr Pro Pro His Glu His 65 70 75 80Ile Cys Leu Ser Asp Glu Leu Pro Pro Gln Gly Ser Pro Val Pro Thr 85 90 95Gln Asp Arg Gly Thr Ser Thr Asp Ser Pro Cys Arg Arg Ser Ala Ala 100 105110 Thr Gln Met Ala Pro Pro Ser Gly Pro Pro Ala Thr Ala Pro Gly Gly 115120 125 Arg Gly His Ser His Arg Asp Arg Ile His Tyr Gln Ala Asp Val Arg130 135 140 Leu Glu Ala Thr Glu Glu Ile Tyr Leu Thr Pro 145 150 155 27amino acids amino acid linear peptide c-Jun JNK-binding domain 5 Tyr SerAsn Pro Lys Ile Leu Lys Gln Ser Met Thr Leu Asn Leu Ala 1 5 10 15 AspPro Val Gly Ser Leu Lys Pro His Leu Arg 20 25 27 amino acids amino acidlinear peptide ATF-2 JNK-binding domain 6 His Leu Ala Val His Lys HisLys His Glu Met Thr Leu Lys Phe Gly 1 5 10 15 Pro Ala Arg Asn Asp SerVal Ile Val Ala Asp 20 25 27 amino acids amino acid linear peptide JIP-1mutant (159-162) G 7 Ser Gly Asp Thr Tyr Arg Pro Lys Arg Pro Thr Gly GlyGly Gly Phe 1 5 10 15 Pro Gln Val Pro Arg Ser Gln Asp Thr Leu Asn 20 2527 amino acids amino acid linear peptide JIP-1 mutant T159G 8 Ser GlyAsp Thr Tyr Arg Pro Lys Arg Pro Thr Gly Leu Asn Leu Phe 1 5 10 15 ProGln Val Pro Arg Ser Gln Asp Thr Leu Asn 20 25 27 amino acids amino acidlinear peptide JIP-1 mutant L160G 9 Ser Gly Asp Thr Tyr Arg Pro Lys ArgPro Thr Thr Gly Asn Leu Phe 1 5 10 15 Pro Gln Val Pro Arg Ser Gln AspThr Leu Asn 20 25 27 amino acids amino acid linear peptide JIP-1 mutantL162G 10 Ser Gly Asp Thr Tyr Arg Pro Lys Arg Pro Thr Thr Leu Asn Gly Phe1 5 10 15 Pro Gln Val Pro Arg Ser Gln Asp Thr Leu Asn 20 25 27 aminoacids amino acid linear peptide JIP-1 mutant K155G 11 Ser Gly Asp ThrTyr Arg Pro Gly Arg Pro Thr Thr Leu Asn Leu Phe 1 5 10 15 Pro Gln ValPro Arg Ser Gln Asp Thr Leu Asn 20 25 27 amino acids amino acid linearpeptide control peptide 12 Asn Ser Leu Gly Thr Asp Asp Thr Gln Tyr SerArg Arg Pro Pro Lys 1 5 10 15 Val Arg Gln Pro Asn Pro Thr Phe Thr LeuLeu 20 25 50 amino acids amino acid linear peptide JIP-1 amino acids491-540 (SH3 domain) 13 Ala Ile Phe Arg Glu Val Pro Arg His Glu Asp GluLeu Glu Leu Glu 1 5 10 15 Val Asp Asp Pro Leu Leu Val Glu Leu Gln AlaGlu Asp Tyr Trp Tyr 20 25 30 Glu Ala Tyr Asn Met Arg Thr Gly Ala Arg GlyVal Phe Pro Ala Tyr 35 40 45 Tyr Ala 50 8 amino acids amino acid linearpeptide Flag eptiope 14 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 76 aminoacids amino acid linear peptide JIP-1 amino acids 127-202 15 Pro Lys AlaGlu Ser Asn Gln Asp Pro Ala Pro Arg Ser Gln Gly Gln 1 5 10 15 Gly ProGly Thr Gly Ser Gly Asp Thr Tyr Arg Pro Lys Arg Pro Thr 20 25 30 Thr LeuAsn Leu Phe Pro Gln Val Pro Arg Ser Gln Asp Thr Leu Asn 35 40 45 Asn AsnSer Leu Gly Lys Lys His Ser Trp Gln Asp Arg Val Ser Arg 50 55 60 Ser SerSer Pro Leu Lys Thr Gly Glu Gln Thr Pro 65 70 75 79 amino acids aminoacid linear peptide JIP-1 amino acids 203-281 16 Pro His Glu His Ile CysLeu Ser Asp Glu Leu Pro Pro Gln Gly Ser 1 5 10 15 Pro Val Pro Thr GlnAsp Arg Gly Thr Ser Thr Asp Ser Pro Cys Arg 20 25 30 Arg Ser Ala Ala ThrGln Met Ala Pro Pro Ser Gly Pro Pro Ala Thr 35 40 45 Ala Pro Gly Gly ArgGly His Ser His Arg Asp Arg Ile His Tyr Gln 50 55 60 Ala Asp Val Arg LeuGlu Ala Thr Glu Glu Ile Tyr Leu Thr Pro 65 70 75 77 amino acids aminoacid linear peptide JIP-1 amino acids 164-240 17 Pro Gln Val Pro Arg SerGln Asp Thr Leu Asn Asn Asn Ser Leu Gly 1 5 10 15 Lys Lys His Ser TrpGln Asp Arg Val Ser Arg Ser Ser Ser Pro Leu 20 25 30 Lys Thr Gly Glu GlnThr Pro Pro His Glu His Ile Cys Leu Ser Asp 35 40 45 Glu Leu Pro Pro GlnGly Ser Pro Val Pro Thr Gln Asp Arg Gly Thr 50 55 60 Ser Thr Asp Ser ProCys Arg Arg Ser Ala Ala Thr Gln 65 70 75 68 amino acids amino acidlinear peptide JIP-1 amino acids 135-202 18 Pro Ala Pro Arg Ser Gln GlyGln Gly Pro Gly Thr Gly Ser Gly Asp 1 5 10 15 Thr Tyr Arg Pro Lys ArgPro Thr Thr Leu Asn Leu Phe Pro Gln Val 20 25 30 Pro Arg Ser Gln Asp ThrLeu Asn Asn Asn Ser Leu Gly Lys Lys His 35 40 45 Ser Trp Gln Asp Arg ValSer Arg Ser Ser Ser Pro Leu Lys Thr Gly 50 55 60 Glu Gln Thr Pro 65 59amino acids amino acid linear peptide JIP-1 amino acids 144-202 19 ProGly Thr Gly Ser Gly Asp Thr Tyr Arg Pro Lys Arg Pro Thr Thr 1 5 10 15Leu Asn Leu Phe Pro Gln Val Pro Arg Ser Gln Asp Thr Leu Asn Asn 20 25 30Asn Ser Leu Gly Lys Lys His Ser Trp Gln Asp Arg Val Ser Arg Ser 35 40 45Ser Ser Pro Leu Lys Thr Gly Glu Gln Thr Pro 50 55 49 amino acids aminoacid linear peptide JIP-1 amino acids 154-202 20 Pro Lys Arg Pro Thr ThrLeu Asn Leu Phe Pro Gln Val Pro Arg Ser 1 5 10 15 Gln Asp Thr Leu AsnAsn Asn Ser Leu Gly Lys Lys His Ser Trp Gln 20 25 30 Asp Arg Val Ser ArgSer Ser Ser Pro Leu Lys Thr Gly Glu Gln Thr 35 40 45 Pro 39 amino acidsamino acid linear peptide JIP-1 amino acids 164-202 21 Pro Gln Val ProArg Ser Gln Asp Thr Leu Asn Asn Asn Ser Leu Gly 1 5 10 15 Lys Lys HisSer Trp Gln Asp Arg Val Ser Arg Ser Ser Ser Pro Leu 20 25 30 Lys Thr GlyGlu Gln Thr Pro 35 17 amino acids amino acid linear peptide JIP-1 aminoacids 127-143 22 Pro Lys Ala Glu Ser Asn Gln Asp Pro Ala Pro Arg Ser GlnGly Gln 1 5 10 15 Gly 27 amino acids amino acid linear peptide JIP-1amino acids 127-153 23 Pro Lys Ala Glu Ser Asn Gln Asp Pro Ala Pro ArgSer Gln Gly Gln 1 5 10 15 Gly Pro Gly Thr Gly Ser Gly Asp Thr Tyr Arg 2025 37 amino acids amino acid linear peptide JIP-1 amino acids 127-163 24Pro Lys Ala Glu Ser Asn Gln Asp Pro Ala Pro Arg Ser Gln Gly Gln 1 5 1015 Gly Pro Gly Thr Gly Ser Gly Asp Thr Tyr Arg Pro Lys Arg Pro Thr 20 2530 Thr Leu Asn Leu Phe 35 20 base pairs nucleic acid single linear DNA6...6 where R at position 6 is A or G 25 GARGARTTYG ARGAYGARGA 20 20base pairs nucleic acid single linear DNA 6...6 where R at position 6 isA or G 26 GGNAARAARC AYAGNTGGCA 20 21 base pairs nucleic acid singlelinear DNA 7...7 where W at position 7 is A or T 27 CATRTTWTANGCYTCWTACC A 21 23 base pairs nucleic acid single linear DNA 6...6 whereY at position 6 is C or T 28 AAYTGYTTKT ARAAYTGYTG RAA 23 33 base pairsnucleic acid single linear RNA 29 GGUAUCGAUA AGCUUGAUAU CGCUGUCCGG AGC33 33 base pairs nucleic acid single linear RNA 30 AGAGGCACUG UCCCAUCCUGGGCCUGUUUC AUG 33

What is claimed is:
 1. A substantially pure JNK-interacting protein 1(JIP-1) polypeptide or a biologically active fragment thereof.
 2. Thepolypeptide of claim 1, wherein said polypeptide is modified byattachment of a hydrophobic moiety, to facilitate uptake of saidpolypeptide by cells.
 3. The polypeptide of claim 2, wherein saidpolypeptide is modified by attachment of a peptide that facilitatesuptake of said polypeptide by cells.
 4. The polypeptide of claim 1,wherein said polypeptide comprises an amino acid sequence correspondingto the JNK-binding domain (JBD; SEQ ID NO:4) of wild type JIP-1.
 5. Thepolypeptide of claim 1, said polypeptide comprising an amino acidsequence substantially identical to SEQ ID NO:1.
 6. The polypeptide ofclaim 1, said polypeptide comprising an amino acid sequencesubstantially identical to a sequence selected from the group consistingSEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:16; SEQID NO:17; SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ IDNO:22; SEQ ID NO:22; SEQ ID NO:23; and SEQ ID NO:24.
 7. The polypeptideof claim 1, wherein said polypeptide is of mammalian origin.
 8. Thepolypeptide of claim 1, wherein said polypeptide is of human origin. 9.Isolated nucleic acid comprising a sequence encoding the polypeptide ofclaim 1, or its complement.
 10. The nucleic acid of claim 9, whereinsaid nucleic acid comprises a sequence encoding a JNK-binding domain.11. The nucleic acid of claim 9, said nucleic acid comprising anucleotide sequence substantially identical to SEQ ID NO:12 or itscomplement.
 12. The nucleic acid of claim 9, said nucleic acid encodingthe amino acid sequence of SEQ ID NO:1.
 13. A genetically engineeredhost cell comprising the nucleic acid of claim
 9. 14. An expressionvector comprising the nucleic acid of claim 9, operably linked to anucleotide sequence regulatory element that controls expression of thenucleotide sequence in a host cell.
 15. The nucleic acid of claim 9,wherein said nucleic acid is of mammalian origin.
 16. The nucleic acidof claim 9, wherein said nucleic acid is of human origin.
 17. A methodof treating a patient having a pathological condition associated withabnormal expression or activity of JNK, or at risk of developing apathological condition associated with abnormal activity or expressionof JNK, said method comprising administering to the patient atherapeutically effective amount of a JIP-1 nucleic acid.
 18. The methodof claim 17, wherein said pathological condition is a neurodegenerativedisease.
 19. The method of claim 17, wherein said neurodegenerativedisease is selected from the group consisting of Parkinson's disease andAlzheimer's disease.
 20. The method of claim 17, wherein saidpathological condition is a blood clot.
 21. The method of claim 17,wherein said condition is stroke.
 22. The method of claim 17, whereinsaid pathological condition is malignancy.
 23. The method of claim 17,wherein said pathological condition is leukemia.
 24. The method of claim23, wherein said leukemia is chronic myelogenous leukemia.
 25. Themethod of claim 17, wherein said pathological condition is an autoimmunedisease.
 26. The method of claim 17, wherein said pathological conditionis inflammation.
 27. A method of treating a patient having apathological condition associated with abnormal expression or activityof JNK, or at risk of developing a pathological condition associatedwith abnormal activity or expression of JNK, said method comprisingadministering to the patient a therapeutically effective amount of aJIP-1 polypeptide.